CA2868161A1 - Anti-hcmv idiotypic antibodies and uses thereof - Google Patents
Anti-hcmv idiotypic antibodies and uses thereof Download PDFInfo
- Publication number
- CA2868161A1 CA2868161A1 CA 2868161 CA2868161A CA2868161A1 CA 2868161 A1 CA2868161 A1 CA 2868161A1 CA 2868161 CA2868161 CA 2868161 CA 2868161 A CA2868161 A CA 2868161A CA 2868161 A1 CA2868161 A1 CA 2868161A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- seq
- hvr
- amino acid
- chain sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 claims abstract description 145
- 241000701024 Human betaherpesvirus 5 Species 0.000 claims abstract description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 150
- 239000000523 sample Substances 0.000 claims description 80
- 239000003153 chemical reaction reagent Substances 0.000 claims description 65
- 239000012472 biological sample Substances 0.000 claims description 56
- 239000011324 bead Substances 0.000 claims description 44
- 238000001514 detection method Methods 0.000 claims description 37
- 210000002966 serum Anatomy 0.000 claims description 36
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 35
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 32
- 238000002965 ELISA Methods 0.000 claims description 29
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 24
- 239000000470 constituent Substances 0.000 claims description 22
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 claims description 21
- 238000000926 separation method Methods 0.000 claims description 17
- 238000011534 incubation Methods 0.000 claims description 16
- 238000004949 mass spectrometry Methods 0.000 claims description 16
- 108010090804 Streptavidin Proteins 0.000 claims description 15
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 14
- 229960002685 biotin Drugs 0.000 claims description 13
- 239000011616 biotin Substances 0.000 claims description 13
- 235000020958 biotin Nutrition 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 10
- 210000002381 plasma Anatomy 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 8
- 238000004587 chromatography analysis Methods 0.000 claims description 7
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 230000005291 magnetic effect Effects 0.000 claims description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 238000003018 immunoassay Methods 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 230000005298 paramagnetic effect Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 68
- 238000003556 assay Methods 0.000 description 64
- 230000027455 binding Effects 0.000 description 44
- 108090000623 proteins and genes Proteins 0.000 description 34
- 238000006467 substitution reaction Methods 0.000 description 31
- 239000000427 antigen Substances 0.000 description 30
- 102000036639 antigens Human genes 0.000 description 30
- 108091007433 antigens Proteins 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 30
- 102000004169 proteins and genes Human genes 0.000 description 30
- 150000007523 nucleic acids Chemical class 0.000 description 29
- 108020004707 nucleic acids Proteins 0.000 description 28
- 102000039446 nucleic acids Human genes 0.000 description 28
- 102000004196 processed proteins & peptides Human genes 0.000 description 25
- 239000000203 mixture Substances 0.000 description 22
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- 235000001014 amino acid Nutrition 0.000 description 21
- 150000002500 ions Chemical class 0.000 description 21
- 239000012491 analyte Substances 0.000 description 19
- 238000003908 quality control method Methods 0.000 description 19
- 125000000539 amino acid group Chemical group 0.000 description 17
- 210000004408 hybridoma Anatomy 0.000 description 17
- 239000000872 buffer Substances 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 238000003780 insertion Methods 0.000 description 15
- 230000037431 insertion Effects 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 14
- 239000013598 vector Substances 0.000 description 14
- 241000701022 Cytomegalovirus Species 0.000 description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 12
- 241000124008 Mammalia Species 0.000 description 12
- 229940098773 bovine serum albumin Drugs 0.000 description 12
- 238000012217 deletion Methods 0.000 description 12
- 230000037430 deletion Effects 0.000 description 12
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 12
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 11
- 238000010790 dilution Methods 0.000 description 11
- 239000012895 dilution Substances 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 11
- 230000035945 sensitivity Effects 0.000 description 11
- 241001529936 Murinae Species 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 10
- 230000004044 response Effects 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 9
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 9
- 238000000132 electrospray ionisation Methods 0.000 description 9
- 239000012071 phase Substances 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 239000011534 wash buffer Substances 0.000 description 9
- 239000012530 fluid Substances 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 206010035226 Plasma cell myeloma Diseases 0.000 description 7
- 210000004602 germ cell Anatomy 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 7
- 201000000050 myeloid neoplasm Diseases 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000011002 quantification Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000012286 ELISA Assay Methods 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 6
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- -1 SEQ ID NOs: 29-34) Chemical compound 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 150000001793 charged compounds Chemical class 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 6
- 102000018358 immunoglobulin Human genes 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 230000004481 post-translational protein modification Effects 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 5
- 230000004075 alteration Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 230000016784 immunoglobulin production Effects 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 229940068977 polysorbate 20 Drugs 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 239000007790 solid phase Substances 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 4
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 206010010356 Congenital anomaly Diseases 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000000306 component Substances 0.000 description 4
- 239000000562 conjugate Substances 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 210000003754 fetus Anatomy 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 229940124452 immunizing agent Drugs 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- 238000004885 tandem mass spectrometry Methods 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 3
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 102000006395 Globulins Human genes 0.000 description 3
- 108010044091 Globulins Proteins 0.000 description 3
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 3
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 108091006629 SLC13A2 Proteins 0.000 description 3
- 230000009824 affinity maturation Effects 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 229940049595 antibody-drug conjugate Drugs 0.000 description 3
- 238000000065 atmospheric pressure chemical ionisation Methods 0.000 description 3
- 239000013060 biological fluid Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 229940127121 immunoconjugate Drugs 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 238000001155 isoelectric focusing Methods 0.000 description 3
- 150000002605 large molecules Chemical class 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 210000002826 placenta Anatomy 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000017854 proteolysis Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- KGLPWQKSKUVKMJ-UHFFFAOYSA-N 2,3-dihydrophthalazine-1,4-dione Chemical class C1=CC=C2C(=O)NNC(=O)C2=C1 KGLPWQKSKUVKMJ-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 2
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 2
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 2
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 2
- 108090000331 Firefly luciferases Proteins 0.000 description 2
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108010015133 Galactose oxidase Proteins 0.000 description 2
- 102000002464 Galactosidases Human genes 0.000 description 2
- 108010093031 Galactosidases Proteins 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 2
- 108010023244 Lactoperoxidase Proteins 0.000 description 2
- 102000045576 Lactoperoxidases Human genes 0.000 description 2
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 2
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 206010036790 Productive cough Diseases 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- 244000000188 Vaccinium ovalifolium Species 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 108010093894 Xanthine oxidase Proteins 0.000 description 2
- 102100033220 Xanthine oxidase Human genes 0.000 description 2
- 238000012867 alanine scanning Methods 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 210000004381 amniotic fluid Anatomy 0.000 description 2
- 230000003466 anti-cipated effect Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 239000010836 blood and blood product Substances 0.000 description 2
- 239000012503 blood component Substances 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 229940125691 blood product Drugs 0.000 description 2
- ZADPBFCGQRWHPN-UHFFFAOYSA-N boronic acid Chemical compound OBO ZADPBFCGQRWHPN-UHFFFAOYSA-N 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000004807 desolvation Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000012156 elution solvent Substances 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 210000001733 follicular fluid Anatomy 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 125000000623 heterocyclic group Chemical group 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000008629 immune suppression Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 229940057428 lactoperoxidase Drugs 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 210000004880 lymph fluid Anatomy 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 108010029942 microperoxidase Proteins 0.000 description 2
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 229910052761 rare earth metal Inorganic materials 0.000 description 2
- 150000002910 rare earth metals Chemical class 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000003802 sputum Anatomy 0.000 description 2
- 208000024794 sputum Diseases 0.000 description 2
- 210000001179 synovial fluid Anatomy 0.000 description 2
- 210000001138 tear Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 2
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- HJBUBXIDMQBSQW-UHFFFAOYSA-N 4-(4-diazoniophenyl)benzenediazonium Chemical compound C1=CC([N+]#N)=CC=C1C1=CC=C([N+]#N)C=C1 HJBUBXIDMQBSQW-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- NLPWSMKACWGINL-UHFFFAOYSA-N 4-azido-2-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(N=[N+]=[N-])C=C1O NLPWSMKACWGINL-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- IPWKGIFRRBGCJO-IMJSIDKUSA-N Ala-Ser Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O IPWKGIFRRBGCJO-IMJSIDKUSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-N 0.000 description 1
- MECFLTFREHAZLH-ACZMJKKPSA-N Asn-Glu-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N MECFLTFREHAZLH-ACZMJKKPSA-N 0.000 description 1
- JHFNSBBHKSZXKB-VKHMYHEASA-N Asp-Gly Chemical compound OC(=O)C[C@H](N)C(=O)NCC(O)=O JHFNSBBHKSZXKB-VKHMYHEASA-N 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010062343 Congenital infection Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 206010048843 Cytomegalovirus chorioretinitis Diseases 0.000 description 1
- 206010011878 Deafness Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 101000935587 Homo sapiens Flavin reductase (NADPH) Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- JPNRPAJITHRXRH-BQBZGAKWSA-N Lys-Asn Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC(N)=O JPNRPAJITHRXRH-BQBZGAKWSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 241000237988 Patellidae Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- KIQUCMUULDXTAZ-HJOGWXRNSA-N Phe-Tyr-Tyr Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O KIQUCMUULDXTAZ-HJOGWXRNSA-N 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 208000037656 Respiratory Sounds Diseases 0.000 description 1
- 206010038910 Retinitis Diseases 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 101000805921 Strongylocentrotus purpuratus Upstream stimulatory factor Proteins 0.000 description 1
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- KHPLUFDSWGDRHD-SLFFLAALSA-N Tyr-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O KHPLUFDSWGDRHD-SLFFLAALSA-N 0.000 description 1
- 101150117115 V gene Proteins 0.000 description 1
- 101000671634 Xenopus borealis Upstream stimulatory factor 1 Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000002313 adhesive film Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000011166 aliquoting Methods 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000611 antibody drug conjugate Substances 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 238000010913 antigen-directed enzyme pro-drug therapy Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 101150070557 artM gene Proteins 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 229960002319 barbital Drugs 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000013626 chemical specie Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 208000001763 cytomegalovirus retinitis Diseases 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000022811 deglycosylation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000004401 flow injection analysis Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036252 glycation Effects 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 230000009643 growth defect Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 230000010370 hearing loss Effects 0.000 description 1
- 231100000888 hearing loss Toxicity 0.000 description 1
- 208000016354 hearing loss disease Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 230000000521 hyperimmunizing effect Effects 0.000 description 1
- 150000002463 imidates Chemical class 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 201000006747 infectious mononucleosis Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000013383 initial experiment Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000011236 particulate material Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 108010054442 polyalanine Proteins 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 206010037833 rales Diseases 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000013391 scatchard analysis Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- AGDSCTQQXMDDCV-UHFFFAOYSA-M sodium;2-iodoacetate Chemical compound [Na+].[O-]C(=O)CI AGDSCTQQXMDDCV-UHFFFAOYSA-M 0.000 description 1
- 238000000935 solvent evaporation Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229940031439 squalene Drugs 0.000 description 1
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- JLEXUIVKURIPFI-UHFFFAOYSA-N tris phosphate Chemical compound OP(O)(O)=O.OCC(N)(CO)CO JLEXUIVKURIPFI-UHFFFAOYSA-N 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 230000007502 viral entry Effects 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4216—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-viral Ig
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/089—Cytomegalovirus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/686—Anti-idiotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/088—Varicella-zoster virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/04—Varicella-zoster virus
- G01N2333/045—Cytomegalovirus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides anti-idiotypic HCMV antibodies as well as methods of using the same.
Description
ANTI-HCMV IDIOTYPIC ANTIBODIES AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to anti-HCMV idiotypic antibodies and methods of using the same, BACKGROUND
Human cytomegalovirus (HCMV) is a 0-herpesvirus and is also known as human herpesvirus-5 (HMI-5). Other species of cytomegalovirus (CMV) exist which infect additional mammals such as murine CMV (MCMV), guinea pig CMV (C.IPCMV), simian CMV
(SCCMV), rhesus CMV (rhCMV) and chimpanzee CMV (CCMV). HCMV is a common herpesvirus that infects nearly 50% of the U.S. population. For the vast majority of human infected individuals, HCMV infection is asymptomatic. However, in conditions of illness, and immune suppression (e,g., HIV infection, drug-induced immune suppression in transplant patients) HCMV
reactivation or primary infection causes a variety of clinical manifestations such as mononucleosis, hepatitis, retinitis, pneumonia, blindness and organ failure.
In addition, in the setting of pregiancy, the acquisition of primary CMV infection, though of little consequence to the mother, can have severe clinical consequences in the developing fetus, Congenital HCMV infection is of particular importance as many children born to mothers infected during pregnancy become infected in Filer and suffer devastating clinical disease. In the United States and Europe, 126,000 women have primary HCMV infection during pregnancy and approximately 40,000 of the babies born to these mothers have congenital infection. In the U.S., I in 750 children are born with or develop disabilities due to HCMV
infection, including:
mental retardation, hearing loss, vision loss, organ defects, and growth defects. Congenital HCMV infection is the most common infectious cause of fetal abnormalities.
After primary infection of a pregnant woman has occurred, there is currently no approved therapy for the prevention or treatment of fetal infection.
In 2005, Nigo and colleagues published a study in which human CMV hyperimmune globulin (HIG) was administered to expectant mothers with primary HCMV
infection (Nigro et aL (2005) New EngL ar. Med. 353:1350-1362). In one arm of the study only I of the 31 infants born to HOVIV-infected mothers were born with disease while 7114 (50%) of children born to untreated women were born with HCMV disease. id.
During pregnancy, HCMV can spread from the infected mother to the fetus via the placenta. The placenta, which anchors the fetus to the uterus, contains specialized epithelial cells, stromal fibroblast cells, endothelial cells, and specialized macrophages. The HCMV viral surface contains various viral glycoprotein complexes that have been shown to be required tbr infection of the specific cell types found in the placenta. A complex of CMV
glycoproteins containing gH/gL and ULI 28, ULt 30 and UL131 (herein referred to as "Complex 1") is specifically required for infection of endothelial cells, epithelial cells and macrophages. A
complex of CMV glycoproteins containing gH/gL and g0 (herein referred to as "Complex H") is specifically required for infection of fibroblasts. HIG has been shown to block viral entry into all four of the placental cell types that are susceptible to HCMV infection, Due to the difficulty of preparing and widely distributing IEG and the reluctance of physicians and the medical community to use human blood products, particularly in pregnant women, it would be most beneficial to create a composition comprising a monoclonal antibody or monoclonal antibodies that could protect fetuses from congenital HCMV
infection. No monoclonal antibody composition to date has been developed for the prevention of maternal-fetal transmission of CMV. Lanzavecchia and Macagno have disclosed naturally-occurring antibodies that were isolated from the immortalized B cells of infected patients that bind to a conformational epitope resulting from the combination of UL130 and IiL131 or a combination of UL128, .UL130 and UL131 that neutralizes CMV transmission (U.S. Patent Publication Nos.
2008./0213265 and 2009/0081230). Shenk and Wang have disclosed antibodies that bind to proteins of Complex I (U.S. Patent No. 7,704,510). Funaro et al. also disclose neutralizing antibodies to CMV in U.S. Patent Publication No. 2010-0040602. Additionally, an anti-gH
monoclonal antibody, NISL-109 was tested in humans in two patient populations, allogenic bone marrow transplant recipients and patients with AIDS and CMV retinitis (Probyski et al., Transplantation 51:1190-1196 (1991); Boeckh et al., Biol. Blood Marrow Transplant. 7:343-351 (2001); and Borucki eta!,, Antiviral Res. 64:103-111 (2004) without success, U.S. Application Serial No. 13/248,998, incorporated by reference herein in its entirety, discloses humanized anti-HCMV monoclonal antibodies. Antibodies disclosed in U.S.
Application No, 13/248,998 were shown to have neutralizing potency comparable to human inununoglobulin from patients infected with HCVM (HIG) for inhibiting infection on fibroblasts, epithelial cells, endothelial cells and macrophages. These antibodies are useful, for example, for the prevention, inhibition and/or treatment of HCMV infection, congenital HCMV
infection and infection of patients through HCMV-infected transplanted tissues.
FIELD OF THE INVENTION
The present invention relates to anti-HCMV idiotypic antibodies and methods of using the same, BACKGROUND
Human cytomegalovirus (HCMV) is a 0-herpesvirus and is also known as human herpesvirus-5 (HMI-5). Other species of cytomegalovirus (CMV) exist which infect additional mammals such as murine CMV (MCMV), guinea pig CMV (C.IPCMV), simian CMV
(SCCMV), rhesus CMV (rhCMV) and chimpanzee CMV (CCMV). HCMV is a common herpesvirus that infects nearly 50% of the U.S. population. For the vast majority of human infected individuals, HCMV infection is asymptomatic. However, in conditions of illness, and immune suppression (e,g., HIV infection, drug-induced immune suppression in transplant patients) HCMV
reactivation or primary infection causes a variety of clinical manifestations such as mononucleosis, hepatitis, retinitis, pneumonia, blindness and organ failure.
In addition, in the setting of pregiancy, the acquisition of primary CMV infection, though of little consequence to the mother, can have severe clinical consequences in the developing fetus, Congenital HCMV infection is of particular importance as many children born to mothers infected during pregnancy become infected in Filer and suffer devastating clinical disease. In the United States and Europe, 126,000 women have primary HCMV infection during pregnancy and approximately 40,000 of the babies born to these mothers have congenital infection. In the U.S., I in 750 children are born with or develop disabilities due to HCMV
infection, including:
mental retardation, hearing loss, vision loss, organ defects, and growth defects. Congenital HCMV infection is the most common infectious cause of fetal abnormalities.
After primary infection of a pregnant woman has occurred, there is currently no approved therapy for the prevention or treatment of fetal infection.
In 2005, Nigo and colleagues published a study in which human CMV hyperimmune globulin (HIG) was administered to expectant mothers with primary HCMV
infection (Nigro et aL (2005) New EngL ar. Med. 353:1350-1362). In one arm of the study only I of the 31 infants born to HOVIV-infected mothers were born with disease while 7114 (50%) of children born to untreated women were born with HCMV disease. id.
During pregnancy, HCMV can spread from the infected mother to the fetus via the placenta. The placenta, which anchors the fetus to the uterus, contains specialized epithelial cells, stromal fibroblast cells, endothelial cells, and specialized macrophages. The HCMV viral surface contains various viral glycoprotein complexes that have been shown to be required tbr infection of the specific cell types found in the placenta. A complex of CMV
glycoproteins containing gH/gL and ULI 28, ULt 30 and UL131 (herein referred to as "Complex 1") is specifically required for infection of endothelial cells, epithelial cells and macrophages. A
complex of CMV glycoproteins containing gH/gL and g0 (herein referred to as "Complex H") is specifically required for infection of fibroblasts. HIG has been shown to block viral entry into all four of the placental cell types that are susceptible to HCMV infection, Due to the difficulty of preparing and widely distributing IEG and the reluctance of physicians and the medical community to use human blood products, particularly in pregnant women, it would be most beneficial to create a composition comprising a monoclonal antibody or monoclonal antibodies that could protect fetuses from congenital HCMV
infection. No monoclonal antibody composition to date has been developed for the prevention of maternal-fetal transmission of CMV. Lanzavecchia and Macagno have disclosed naturally-occurring antibodies that were isolated from the immortalized B cells of infected patients that bind to a conformational epitope resulting from the combination of UL130 and IiL131 or a combination of UL128, .UL130 and UL131 that neutralizes CMV transmission (U.S. Patent Publication Nos.
2008./0213265 and 2009/0081230). Shenk and Wang have disclosed antibodies that bind to proteins of Complex I (U.S. Patent No. 7,704,510). Funaro et al. also disclose neutralizing antibodies to CMV in U.S. Patent Publication No. 2010-0040602. Additionally, an anti-gH
monoclonal antibody, NISL-109 was tested in humans in two patient populations, allogenic bone marrow transplant recipients and patients with AIDS and CMV retinitis (Probyski et al., Transplantation 51:1190-1196 (1991); Boeckh et al., Biol. Blood Marrow Transplant. 7:343-351 (2001); and Borucki eta!,, Antiviral Res. 64:103-111 (2004) without success, U.S. Application Serial No. 13/248,998, incorporated by reference herein in its entirety, discloses humanized anti-HCMV monoclonal antibodies. Antibodies disclosed in U.S.
Application No, 13/248,998 were shown to have neutralizing potency comparable to human inununoglobulin from patients infected with HCVM (HIG) for inhibiting infection on fibroblasts, epithelial cells, endothelial cells and macrophages. These antibodies are useful, for example, for the prevention, inhibition and/or treatment of HCMV infection, congenital HCMV
infection and infection of patients through HCMV-infected transplanted tissues.
There is a need in the art to detect therapeutic humanized monoclonal antibodies to HCMV in biological samples and/or clinical samples without also detecting other antibodies directed or not directed to HCMV (e.g., endogenous immunoglobulins). The invention provides anti-idiotypic antibodies that specifically detect certain anti-HCMV
antibodies. These antibodies are useful, for example, in pharmacokinetic (PK) and pharmacodynamic studies and for the quantification and monitoring of therapeutic anti-HCMV antibodies in patients.
SUMMARY
The invention provides isolated anti-idiotic antibodies which specifically bind to anti-HCMV monoclonal antibodies. In one embodiment, the invention provides an isolated anti-idiotypic antibody that specifically binds to an anti-HCMV
antibody comprising the heavy, chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID
NO: 2 or to an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain sequence of SEQ ID NO: 2. In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV antibody comprising all six HAIRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2, In some embodiments, the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and FIVR-H3) and three light chain hyperwuiable regions (HVR-L1. HVR-L2 and IIVR-L3), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ
ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) HVR-LI comprises the amino acid sequence of SEQ ID NO: 16; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17;
and (f) IIVR-L3 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV antibody comprising all six HVRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments, the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-111, HVR-H2 and IIVR-143) and three light chain hypervariable regions (FIVR-1,1, HVR-L2 and FIVR-L3), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ
ID NO: 19;
(1) HVII-H2 comprises the amino acid sequence of SEQ ID NO: 20; (c) IIVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22; (e) HVR-1,2 comprises the amino acid sequence of SEQ ID NO: 23;
and (f) IIVR-L3 comprises the amino acid sequence of SEQ ID NO: 24, In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 11.
In some embodiments, the anti-idiotypic antibody specifically binds to at least one IIVR
of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2. In some embodiments, the anti-idiot3ipic antibody specifically binds to at least one IIVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In some embodiments, the anti-idiotypic antibody specifically binds to HVR-H2 (SEQ ID NO: 36) of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID
NO: 4, In some embodiments, the anti-idiotypic antibody binds to an epitope comprised within an amino acid amino acid sequence selected from SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27 and SEQ ID NO: 28. In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO:
or SEQ ID NO: 26, In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO:
27 or SEQ
ID NO: 28.
In some embodiments, any one of the above anti-idiotypic antibodies is conjugated to a 25 detectable label. In some embodiments, any one of the above anti-idiot:,,,,pic antibodies is conjugated to biotin.
The invention further provides methods of detection using the anti-idiotypic antibodies of the invention. In one embodiment, the invention provides an enzyme-linked immunosorbent assay (ELBA) method for specifically detecting in a biological sample an antibody of interest comprising (a) contacting and incubating the biological sample with a capture reagent, wherein the capture reagent is the anti-idiotypic antibody of claim 1, so as to bind any of the antibody of interest present in the sample, and (h) contacting the capture reagent, and hence any bound antibody of interest, with a detectable antibody that binds to the antibody of interest, and measuring the level of the antibody of interest bound to the anti-idiotypic antibody using a detection means for the detectable antibody, wherein the antibody of interest is selected from (a) a first anti-HCIVIV antibody comprising the heavy chain sequence of SEQ ID NO:
I and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
In some embodiments of the method, the capture reagent is immobilized to a solid support and the method further comprises the step of separating the biological sample from the immobilized capture reagent bound to any of the antibody of interest present, In some embodiments, the immobilized capture reagent is coated on a microtiter plate.
In some embodiments, the immobilized capture reagent is conjugated to biotin and bound to a streptavidin coated microtiter plate.
In some embodiments of the method, the detectable antibody is an antibody from a non-human species that binds to human antibodies. In some embodiments, the detectable antibody is a mouse anti-huigG Fcy antibody.
In some embodiments of the method, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is conjugated to horseradish peroxidase. In some embodiments, the detectable antibody is detected by a fluorimetric or calorimetric reagent.
The invention further provides a method for specifically detecting in a biological sample an antibody of interest comprising: (a) contacting and incubating the biological sample with an anti-idiotypic antibody that specifically binds to the antibody of interest;
(b) contacting and incubating the sample with immunoaffinity beads that bind to the anti-idiotypic antibody; (c) eluting the antibody of interest; (d) applying the eluted antibody of interest to a separation media to effect separation of more than one sample constituent wherein a separated sample constituent comprises the antibody of interest or a fragment or signature peptide thereof;
and (e) establishing the mass to charge ratio of one or more separated sample constituents by mass spectrometry, wherein the antibody of interest is selected from (a) a first anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID
NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
In some embodiments, the method further comprises treating the biological sample with a protease after incubation with the immunoaffmity beads and prior to or after eluting the antibody of interest. In some embodiments, the protease is tr3õpsin, In some embodiments of the method, the anti-idiotypic antibody is biotinylated. In some embodiments, the anti-idiotypic antibody binds to streptavidin coated paramapetic immunoaffinity beads.
In some embodiments of the method, the anti-idiotypic antibodies are bound to the immunoaffinity beads prior to contact and incubation with the biological sample.
In some embodiments of the method, the immunoaffinity bead is a magnetic bead.
In some embodiments of the method, the separation media is a chromatography support.
In various embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
In various embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2 and the anti-idiotypic antibody comprises three heavy chain hypervariable regions (IIVR-H1 IIVR-142 and HVR-113) and three light chain .hypervariable regions (IIVR-L1, HVR-L2 and HVR-L3), wherein: (a) IIVR-H1 comprises the amino acid sequence of SEQ ID NO: 13; (h) HVR-H2 comprises the amino acid sequence of SEQ
ID NO:
14; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:
18. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7.
In various embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4 and the anti-idiot:rale antibody comprises three heavy chain hypervariable regions (HVR-H1, FIVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19; (h) HVR-H2 comprises the amino acid sequence of SEQ
ID NO:
20; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:
24. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID
NO: 9 and the light chain sequence of SEQ ID NO; 11.
.6 In some embodiments of any of the methods disclosed above, the the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I
and the light chain sequence of SEQ ID NO: 2 and the anti-idotypic antibody binds to at least one HVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain sequence of SEQ ID NO: 2.
In some embodiments of any of the methods disclosed above, the the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idotypic antibody binds to at least one HVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In some embodiments, the anti-idiotniic antibody specifically binds to HVR-H2 (SEQ ID NO: 36) of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idotypic antibody binds to an epitope on the anti-HCMV antibody that is comprised within an amino acid sequence selected from SEQ ID NO: 25 or SEQ ID NO: 26.
In some embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4 and the anti-idiotypic antibody binds to an epitope on the anti-HCMV antibody that is comprised within an amino acid amino acid sequence selected from SEQ
ID NO: 27 or SEQ ID NO: 28.
In some embodiments of any of the methods disclosed above, the antibody of interest is a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I
and the light chain sequence of SEQ ID NO: 3 and (b) a second anti -HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4.
In some embodiments of any of the methods disclosed above, the anti-idiotypic antibody binds to the antibody of interest and not to at least one other anti-HCMV
antibody in the sample.
In various embodiments of any of the methods disclosed above, the biological sample is isolated from a human subject. In some embodiments, the the human subject has been treated with an anti-HCMV antibody selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain sequence of SEQ ID
NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
'7 In some embodiments of any of the methods disclosed above, the method further comprises using a standard curve to determine the level of the antibody of interest compared to a known level.
In some embodiments of any of the methods disclosed above, the biological sample is blood, plasma or serum. In some embodiments, the sample is serum.
The invention further provides for a kit. In an embodiment, the invention provides an immunoassay kit for specifically detecting in a biological sample an antibody of interest selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ
ID NO: I and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO:
4; and (c) combination thereof, the kit comprising: (a) a container containing, as a capture reagent, an anti-idiotypic antibody that specifically binds to the antibody of interest; (1)) a container containing a detectable antibody that binds to the antibody of interest; and (c) instructions for detecting said antibody of interest. in some embodiments, the kit is useful in an ELISA
method for detecting the antibody of interest.
In some embodiments, the kit further comprises a solid support for the capture reagent. In some embodiments, the capture reagent is immobilized on the solid support. In some embodiments, the capture reagent is coated on a microliter plate.
In various embodiments, the anti-idiotypic antibody is one or more of any of the anti-antibodies disclosed above. In some embodiments, the anti-idiotypic antibody is selected from (a) a first anti-idiotypic antibody comprising the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7; (b) a second anti-idiotypic antibody comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO:
11; and (c) a combination thereof In some embodiments, the anti-idiotypic antibody specifically binds to an anti-HCMV
antibody comprising at least one heavy chain hypervariable region selected from the group consisting of NO: 13-24. hi one other embodiment, the antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-112 and HVR-H3) and three light chain hypervariable regions (HVR-L1, IIVR-L2 and IIVR-1.3), wherein: (a) HVR-Hl comprises the amino acid sequence of SEQ ID NO: 13; (b) HYR-H2 comprises the amino acid sequence of SEQ
ID NO:
14; (c) IIVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) HVR-IL1 comprises the amino acid sequence of SEQ ID NO: 16; (e) .HVR-1.2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-1,3 comprises the amino acid sequence of SEQ ID NO:
18. In another embodiment, the antibody comprises three heavy chain hypervariable regions (HVR-111, HVR-H2 and HVR-F13) and three light chain hyper-variable regions (HVR-L1, HVR-1.2 and EIVR-L3), wherein: (a) HVR-1-11 comprises the amino acid sequence of SEQ ID
NO: 19; (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22; (e) HVR-1.2 comprises the amino acid sequence of SEQ ID NO: 23;
and (0 HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
The invention also provides isolated nucleic acid encoding the anti-idiotypic HCMV
antibodies of the invention. The invention also provides host cells comprising the nucleic acid encoding such antibodies. The invention further provides a method of producing an antibody comprising culturing the host cells containing the nucleic acid encoding the antibody so that the antibody is produced. The method may further comprise recovering the antibody from the host cell.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows an amino acid sequence alignment of the heavy chain variable region (VH) of murine inAb 4.25B10,15 (SEQ ID NO: 5) with the mouse germline heavy chain variable domain region IGHV1-54*03 (SEQ ID NO: 6). The hypervariable regions (HVRs) are boxed.
Amino acid residues that differ from the mouse germline sequence are highlighted.
Figure 2 shows an amino acid sequence alignment of the light chain variable region (XL) of murine mAb 4,25B10.15 (SEQ ID NO: 7) with the mouse germline heavy chain variable domain region IGKV5-39*01 (SEQ ID NO: 8). The amino acids are numbered according to Kabat numbering. The hypervariable regions (HVRs) are boxed.
Figure 3 shows an amino acid sequence alignment of the heavy chain variable region (VH) of murine inAb 1.9E1.1 (SEQ ID NO: 9) with the mouse germline heavy chain variable domain region IGHV1-50*01 (SEQ ID NO: 10). The hypervariable regions (HVRs) are boxed.
Amino acid residues that differ from the mouse germline sequence are highlighted.
Figure 4 shows an amino acid sequence alignment of the light chain variable region (VL) of murine mAb 1.9E1,1 (SEQ ID NO: 11) with the mouse germline heavy chain variable domain region IGLVI*01 (SEQ ID NO: 12). The amino acids are numbered according to Kabat numbering. The hypervariable regions (HVRs) are boxed. Amino acid residues that differ from the mouse germline sequence are highlighted.
Figure 5 shows an anti-HCMV PK ELISA format whereby a biotin-conjugated anti-HCMV idiotypic antibody (e.g. 1.9E1 .1) binds to a streptavidin coated plate, and to a therapeutic anti-HCMV antibody in solution, The complex is then bound by a mouse anti-human IgG Fey antibody conjugated to HRP for chemiluminescent detection.
Figure 6 shows the binding activity of various purified anti-idiotypic antibodies to the anti-HCMV antibodies anti-CI and anti-gH as determined using the anti-HCMV PK
ELISA
described in Example 2.
Figure 7 shows the recovery of individual serum samples spiked with the anti-HCMV
antibody anti-gH using anti-idiotypic monoclonal antibody 1.9E1.1 as the capture antibody in the anti-HCMV PK ELISA assay described in Example 2.
Figure 8 shows an anti-HCMV PK ELISA format whereby an anti-HCMV idiotypic antibody (e.g. 4.25B10.15) is immobilized on a solid support for binding to a therapeutic anti-HCMV antibody. The complex is then bound by a mouse anti-human IgG Fey antibody conjugated to HRP for chemiluminescent detection.
Figure 9 shows the recovery of individual serum samples spiked with the anti-HCMV
antibody anti-CI using anti-idiot3pic monoclonal antibody 4.25B10.15 or 4.23F9.5 as the capture antibody in the anti-HCMV PK ELISA described in Example 3. Anti-CI was spiked at either at 4 1.1g/mL (high) or 0.5 p,g/ML (low) for the pool sample and samples 1-4; at either 2 ptglmi...
(high) or 0.6 tug/mi., (low) for sample 5, and at either 7,62 p.g/mL (high) or 0.476 (low) for samples 6 and 7.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I, DEFINITIONS
"Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a I :1 interaction between members of a binding pair (e.g., antibody and antigen).
The affinity of a molecule X fbr its partner Y can generally be represented by the dissociation constant (K(i). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
t-(y An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab`)2;
diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG,õ
IgG3, IgG4, IgA I, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, a, 7, and IA, respectively.
The term "detecting" is used in the broadest sense to include both qualitative and quantitative measurements of a target molecule. In one aspect, the detecting method as described herein is used to identify the mere presence of the antibody of interest in a biological sample. In another aspect, the method is used to test whether the antibody of interest in a sample is present at a detectable level. In yet another aspect, the method can be used to quantify the amount of the antibody of interest in a sample and further to compare the antibody levels from different samples.
The term "biological sample" refers to any biological substance that may contain an antibody of interest. A sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, itreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, and other constituents of the body that may contain the antibody of interest. In various embodiments, the sample is a body sample from any animal. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject. In some embodiments, the biological sample is from clinical patients or patients treated with a therapeutic anti-HCMV antibody or antibodies. In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient.
The term "capture reagent" or "coat antibody" refers to an anti-idiotypic antibody or mixture of such antibodies that bind an idiotype of the antibody of interest and are capable of binding and capturing the antibody of interest in a biological sample such that under suitable conditions, the complex of capture reagent and antibody of interest can be separated from the rest of the sample.
An "anti-idiotypic antibody," as used herein, is an antibody that binds to the VH andior V. domain of the cognate antibody, in this case the antibody of interest.
Typically, such anti-idiotypic antibodies are prepared by immunizing a mammal such as a mouse with the antibody of interest and producing a hybridoma and selecting from the panel of antibodies derived from the hybridoma those antibodies that give the cleanest signal in the assay, whether for the capture reagent or the detectable antibody. Typically, the capture reagent is immobilized or immobilizable. Such anti-icliotypic antibodies are monoclonal antibodies and can be for example, rodent antibodies such as murine or rat antibodies.
The terms "anti-Complex I antibody," "anti-CI antibody" or "anti-CI," as used herein, refers to an anti-HCMV antibody comprising the heavy chain variable domain of SEQ ID NO: I
and the light chain variable domain of SEQ ID NO: 2 or an antibody comprising at least one HVR region, as shown below:
EVQLVQSGAEVKKPGASVKVS CKAS GYTFTNYGMNWVRQAPGQGLEWI
GWINTYTGEPTYADDFKGRVT I TRDTS TS TAYLE LS SLRS EDTAVYYC
AR SWYYITS NYWYFDVIIGQG T LVT VS S (SEQ ID NO: 1) The bold, underlined sequences correspond to HVR-HI (SEQ ID NO: 29), HVR-H2 (SEQ ID NO: 30) and HVR-H3 (SEQ ID NO: 31) of SEQ ID NO: 1.
SVLTQS PSASAS LGASVKLTCTLS SQRSTYT I EWYQQQPGKGPRYLMK
LKKDGSHSTGDGI PDRE S GS S SGADRYLT I SNLQS EDEADYYCGVGDT
IKEQFVYVFGGGT KLTVLG (SEQ ID NO: 2) The bold, underlined sequences correspond to HVR-L1 (SEQ ID NO: 32), HVR-L2 (SEQ ID NO: 33) and HVR-L3 (SEQ ID NO: 34) of SEQ ID NO: 2.
The term "anti-gH antibody," or "anti-gH," as used herein, refers to an anti-HCMV
antibody comprising the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4 or an antibody comprising at least one HVR
region, as shown below.
EEQVLE SOGGLATKPGGSLRLS CAA'S GFTF SPYSVFWVRQAPGRGLEWV
SS INSNSRYKYYADSVKGR.FT I SRDNAENS I FLQMNSLRAEDTAVYYC
ARDRSYYAFSSGSLSDYYYGLDVWGQGTLVTVSS (SEQ ID NO: 3) The bold, underlined sequences correspond to HVR-Hl (SEQ ID NO: 35), HVR-H2 (SEQ ID NO: 36) and HVR-H3 (SEQ ID NO: 37) of SEQ ID NO: 3.
DIVMTQS PLS LSVTPGE PAS IS CRS SQSLLHTNGYNYLDWYVQKPGQS
PQLL ThASNRAS GVPDRF SGS GS GTD FMK SRVETEDVGVYYCMQA
LQIPIZTFGQGTKVEIK (SEQ ID NO: 4) The bold, underlined sequences correspond to IIVR-1.,1 (SEQ ID NO: 38), HVR-1,2 (SEQ ID NO: 39) and HVR-L3 (SEQ ID NO: 40) of SEQ ID NO: 4.
An "anti-CI idiotypic antibody," as used herein, is one that specifically binds to an anti-Cl monoclonal antibody having the heavy chain variable domain sequence of SEQ
ID NO: 1 and the light chain variable domain sequence of SEQ ID NO: 2, or to an anti-C1 antibody comprising all six hypervariable regions of an anti-CI monoclonal antibody having the heavy chain variable domain sequence of SEQ ID NO: I and the light chain variable domain sequence of SEQ ID NO:
2 (e.g., SEQ ID NOs: 29-34), with sufficient specificity and affinity to be useful in detection of anti-CI, An "anti-gH idiotypic antibody," as used herein, is one that specifically binds to an anti-gH monoclonal antibody having the heavy chain variable domain sequence of SEQ
ID NO: 3 and the light chain variable domain sequence of SEQ ID NO: 4, or to an anti-gH
antibody comprising all six hypervariable regions of an anti-gIi monoclonal antibody having the heavy chain variable domain sequence of SEQ ID NO: 3 and the light chain variable domain sequence of SEQ ID NO:
4 (e.g,, SEQ ID NOs: 35-40), with sufficient specificity and affinity to be useful in detection of anti-gH.
The term "detectable antibody" refers to an antibody that binds the antibody of interest and is capable of being detected either directly through a label amplified by a detection means, or indirectly through, e.g., another antibody that is labeled. In some embodiments, the detectable antibody is an antibody from a non-human species that binds to human antibodies. In some embodiments, the detectable antibody is an anti-idiotypic antibody or mixture of such antibodies that bind an idiotype of the antibody of interest. For direct labeling, the antibody is typically conjugated to a moiety that is detectable by some means. In some embodiments, the detectable antibody is conjugated to horseradish peroxidase.
The tenu "detection means" refers to a moiety or technique used to detect the presence of the detectable antibody through signal reporting that is then read out in the assay herein. It includes reagents that amplify the immobilized label such as the label captured onto a microliter plate.
The term "Fe region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fe regions and variant Fe regions. In one embodiment, a human IgG
heavy chain Fe region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fe region may or may not be present.
Unless otherwise specified herein, numbering of amino acid residues in the Pc region or constant region is according to the :EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR
domains: FR!, HU, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H (L1)-FR2-H2(1L2)-FR3-H3(L3)-FR4.
The terms "frill length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Pc region as defined herein.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transfbrmants" and transformed cells,"
which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VI, or VH
framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NTH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et aL supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.
A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the FIVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e,g,, a non-human antibody, refers to an antibody that has undergone humanization.
The term "hypervariable region" or "HVR," as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (LI, L2, L3). IIVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complernentarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI ), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, sf. Mot'.
Biol. 196:901-917 (1987),) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-HI, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of Ll , 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3, (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs,"
which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H I, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of Li, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3. (See Almago and Fransson, Front. Biosci.
13:1619-1633 (2008)) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al, supra.
An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
In certain embodiments, the individual or subject is a human.
An "infant" as used herein, refers to an individual or subject ranging in age from birth to not more than about one year and includes infants from 0 to about 12 months.
An "isolated" antibody is one which has been separated from a component of its natural environment, In some embodiments, an antibody is purified to greater than 95%
or 99% purity as determined by, fbr example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), 1.5.
capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase IIPLC). For review of methods for assessment of antibody purity, see, e.g., Flatrnan et al., J. Chromatogr. B
848:79-87 (2007), An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
"Isolated nucleic acid encoding an anti-idiotypic antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or frapnents thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a.
population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin such methods and other exemplary methods for making monoclonal antibodies being described herein.
A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation, "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VT), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain, The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications andior warnings concerning the use of such therapeutic products.
"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the Skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Ivlegalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code, The ALIGN-2 program should be compiled for use on a UNIX
operating system, including digital 'UNIX V4.0DõAll sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the %
amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A
that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y
is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
A "signature peptide" of an anti-HCMV antibody refers to a proteolytic peptide (e.g., a tryptic peptide) that is exclusively present in one antibody isotype. For example, an anti-CI
signature peptide may be a tryptic peptide that is exclusively present in an anti-HCMV antibody comprising the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 2. In a further example, an anti-gH signature peptide may be a tryptic peptide that is exlcusively present in an antibody an anti-HCMV antibody comprising the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ 11) NO: 4, The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervatiable regions (HVRs). (See, e.g,, Kindt et al, Kuby Immunology, 6th. ed., W.H.
Freeman and Co., page 91 (2007)) A single VII or VI, domain may be sufficient to confer antigen-binding specificity, Furthermore, antibodies that bind a particular antigen may be isolated using a VII or VI, domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains; respectively. See, e.g., Portolano et al., .1.
Inumenol.
150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."
H. COMPOSITIONS AND METHODS
In one aspect, the invention provides anti-idiotypic antibodies that specifically bind to the humanized anti-HCMV monoclonal antibodies anti-CI and anti-gH. In certain embodiments, anti-idiotypic antibodies that bind to anti-CI are provided. In certain embodiments, antibodies that bind to anti-gH are provided. Antibodies of the invention are useful, e.g., for the detection and/or quantification of anti-CI and anti-gH in biological samples, for example, in clinical samples.
Aõ Exemplary .Antit-Idiotypie Antibodies In one aspect, the invention provides isolated anti-idiotypic antibodies that bind to anti-HCMV antibodies anti-CI or anti-g-1 with sufficient specificity and affinity to be useful in detection of anti-CI and anti-gH.
In certain embodiments, an anti -idiotypic antibody binds to an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID
NO: 2. In certain embodiments, an an anti-idiotypic antibody binds to an anti-HCMV antibody comprising all six HVRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 201n certain embodiments, an anti-idiotypic antibody binds to an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In certain embodiments, an an anti-idiotypic antibody binds to an anti-HCMV antibody comprising all six HVRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments, the anti-idiot:vpic antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and IIVR-H3) and three light chain hypervariable regions (HVR-1,1, HVR-12 and HVR-L3), wherein:
(a) IIVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-LI comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ. ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
In some embodiments, the anti-idiotypic antibody comprises three heavy chain hypervari able regions (HVR-H I, HVR-H2 and HVR-H3) and three light chain hypervatiable regions (HVR-L1, HVR-1,2 and IIVR-L3), wherein:
(a) HVR-Hl comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) FIVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) IIVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-1,2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 11..
In another aspect, an anti-CI idiotypic antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-CI idiotypic antibody comprising that sequence retains the ability to bind to anti-CI, In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 5. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-CI idiotypic antibody comprises the VII sequence in SEQ ID NO: 5 including post-translational modifications of that sequence. In a particular embodiment, the VII comprises one, two or three 1-1VR,s selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO: 13, (b) .HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) IIVR-H3 comprising the amino acid sequence of SEQ ID NO: 15 In another aspect, an anti-CI idiotypic antibody is provided, wherein the antibody comprises a light chain variable domain (VIõ) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.
In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e,g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C1 idiotypic antibody comprising that sequence retains the ability to bind to anti-Ch In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 7. in certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs in the FRs).
Optionally, the anti-CI idiotypic antibody comprises the VL sequence in SEQ ID
NO: 7, including post-translational modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:
17; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18.
In another aspect, an anti-CI idiotypic antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH
and VL
sequences in SEQ ID NO: 5 and SEQ ID NO: 7, respectively, including post-translational modifications of those sequences.
In another aspect, an anti-gH idiotypic antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-gH idiotypic antibody comprising that sequence retains the ability to bind to anti-gH. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 9. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (ix., in the FRs).
Optionally, the anti-gH idiotypic antibody comprises the VH sequence in SEQ ID NO: 9 including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) IFIVR-H1 comprising the amino acid sequence of SEQ ID
NO: 19, (b) IIVR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-H3 comprising the amino acid sequence of SE() ID NO: 21.
In another aspect, an anti-gli idiotypic antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:
11. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-gH
idiotypic antibody comprising that sequence retains the ability to bind to anti-gH. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ
ID NO: 11. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the FIFV.Rs (i.e., in the FRs), Optionally, the anti-gH idiotypic antibody comprises the VL sequence in SEQ ID NO: II, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L I
comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:
24.
In another aspect, an anti-g1-1 idiotypic antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH
and VL
sequences in SEQ ID NO: 9 and SEQ ID NO: 11, respectively, including post-translational modifications of those sequences.
In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-HCMV idiotypic antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-CI idiotypic antibody comprising a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 7. in certain embodiments, an antibody is provided that binds to the same epitope as an anti-gH idiotypic antibody comprising a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: I. In certain.
embodiments, an antibody is provided that specifically binds to at least one HVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain.
sequence of SEQ ID NO: 2. In certain embodiments, an antibody is provided that specifically binds to at least one IIVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4, In certain embodiments, an antibody is provided that specifically binds to HVR-H2 (SEQ ID NO: 36) of an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In certain embodiments, an antibody is provided that binds to an epitope comprised within an amino acid amino acid sequence selected from SEQ ID NO:
25, SEQ ID
NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO: 25 orSEQ ID NO: 26. In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO:
27 or SEQ ID NO: 28.
In a further aspect of the invention, an anti-HCMV idiotypic antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, an anti-HCMV idiotypic antibody is an antibody fraginent, e.g., a Fv, Fab, Fab', scFv, diabody, or P(a1:02 fragment, In another embodiment, the antibody is a full length antibody, e.g., an intact .1gG1 antibody or other antibody class or isotype as defined herein, A. Antibody Production A description follows as to exemplary techniques for the production of the anti-idiotypic antibodies used in accordance with the present invention.
1. Polvelonal Antibodies The antibodies of the invention may comprise polyclonal antibodies, Methods of preparing polycional antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include anti-CI, anti-gH, an antigen binding fragment thereof, or fusion proteins thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocrmin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation, The mammal can then be bled, and the serum assayed for anti-idiotypic antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus.
2, Monoclonal Antibodies The antibodies of the invention may alternatively be monoclonal antibodies.
Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e/g/ U.S.
Patent No.
4,816,567).
In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myelorria cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp,59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (11GPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfiised parental cells, Myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-Il mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, .S, Immunol.õ
133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp, 51-63 (Marcel Dekker, Inc., New York, 1987)), Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem,, 107:220 (1980).
Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp,59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-1`,AEM
or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.gõ by i,p. injection of the cells into mice.
The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, fur example, affinity chromatography (e.g.õ using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of =rine antibodies). The hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coil cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or rnyeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Cum Opinion in Immunol., 5:256-262 (1993) and Phickthun, Irnmunol, Revs.
130:151-188 (1992).
In a further embodiment, monoclonal antibodies or antibody fragments can be isolated.
from antibody phage libraries generated using the techniques described in McCafferty et al,, Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.
Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Biorrechnology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridorna techniques for isolation of monoclonal antibodies.
In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened for against the desired antigen. Clones expressing Fv .fraguients capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.
Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and .VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev, Immunol., 12: 433-455 (1994).
Repertoires of VH and VL genes can be separately cloned by polyrnerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev, Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., E,1\11B0 1, 12: 725-734 (1993).
Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Screening of the libraries can be accomplished by various techniques known in the art.
For example, anti-CI or anti-gH can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries.
The selection of antibodies with slow dissociation kinetics (and good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al.. Biotechnol.õ 10: 779-783 (1992).
Any of the anti-idiotypic antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-idiotypic antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD
(1991), vols. 1-3.
3. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
a) Substitution, insertion, and Deletion Variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs.
Conservative substitutions are Shown in Table 1 under the heading of "conservative substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g,, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
TABLE
Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; He Val Arg (R) Lys; Gin; Mn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Gin; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Giu Mn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; .Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Lou _________________________ Norleucine Leu (L) .Norleucine; lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin: Asn Arg Met (M) Leu; Phe; lie Len Phe (F) Try; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser TrP (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Lett .Norleueine Amino acids may be grou.ped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Len, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asti, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
antibodies. These antibodies are useful, for example, in pharmacokinetic (PK) and pharmacodynamic studies and for the quantification and monitoring of therapeutic anti-HCMV antibodies in patients.
SUMMARY
The invention provides isolated anti-idiotic antibodies which specifically bind to anti-HCMV monoclonal antibodies. In one embodiment, the invention provides an isolated anti-idiotypic antibody that specifically binds to an anti-HCMV
antibody comprising the heavy, chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID
NO: 2 or to an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain sequence of SEQ ID NO: 2. In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV antibody comprising all six HAIRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2, In some embodiments, the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and FIVR-H3) and three light chain hyperwuiable regions (HVR-L1. HVR-L2 and IIVR-L3), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ
ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) HVR-LI comprises the amino acid sequence of SEQ ID NO: 16; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17;
and (f) IIVR-L3 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In some embodiments, the anti-idotypic antibody specifically binds to an anti-HCMV antibody comprising all six HVRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments, the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-111, HVR-H2 and IIVR-143) and three light chain hypervariable regions (FIVR-1,1, HVR-L2 and FIVR-L3), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ
ID NO: 19;
(1) HVII-H2 comprises the amino acid sequence of SEQ ID NO: 20; (c) IIVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22; (e) HVR-1,2 comprises the amino acid sequence of SEQ ID NO: 23;
and (f) IIVR-L3 comprises the amino acid sequence of SEQ ID NO: 24, In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 11.
In some embodiments, the anti-idiotypic antibody specifically binds to at least one IIVR
of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2. In some embodiments, the anti-idiot3ipic antibody specifically binds to at least one IIVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In some embodiments, the anti-idiotypic antibody specifically binds to HVR-H2 (SEQ ID NO: 36) of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID
NO: 4, In some embodiments, the anti-idiotypic antibody binds to an epitope comprised within an amino acid amino acid sequence selected from SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27 and SEQ ID NO: 28. In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO:
or SEQ ID NO: 26, In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO:
27 or SEQ
ID NO: 28.
In some embodiments, any one of the above anti-idiotypic antibodies is conjugated to a 25 detectable label. In some embodiments, any one of the above anti-idiot:,,,,pic antibodies is conjugated to biotin.
The invention further provides methods of detection using the anti-idiotypic antibodies of the invention. In one embodiment, the invention provides an enzyme-linked immunosorbent assay (ELBA) method for specifically detecting in a biological sample an antibody of interest comprising (a) contacting and incubating the biological sample with a capture reagent, wherein the capture reagent is the anti-idiotypic antibody of claim 1, so as to bind any of the antibody of interest present in the sample, and (h) contacting the capture reagent, and hence any bound antibody of interest, with a detectable antibody that binds to the antibody of interest, and measuring the level of the antibody of interest bound to the anti-idiotypic antibody using a detection means for the detectable antibody, wherein the antibody of interest is selected from (a) a first anti-HCIVIV antibody comprising the heavy chain sequence of SEQ ID NO:
I and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
In some embodiments of the method, the capture reagent is immobilized to a solid support and the method further comprises the step of separating the biological sample from the immobilized capture reagent bound to any of the antibody of interest present, In some embodiments, the immobilized capture reagent is coated on a microtiter plate.
In some embodiments, the immobilized capture reagent is conjugated to biotin and bound to a streptavidin coated microtiter plate.
In some embodiments of the method, the detectable antibody is an antibody from a non-human species that binds to human antibodies. In some embodiments, the detectable antibody is a mouse anti-huigG Fcy antibody.
In some embodiments of the method, the detectable antibody is directly detectable. In some embodiments, the detectable antibody is conjugated to horseradish peroxidase. In some embodiments, the detectable antibody is detected by a fluorimetric or calorimetric reagent.
The invention further provides a method for specifically detecting in a biological sample an antibody of interest comprising: (a) contacting and incubating the biological sample with an anti-idiotypic antibody that specifically binds to the antibody of interest;
(b) contacting and incubating the sample with immunoaffinity beads that bind to the anti-idiotypic antibody; (c) eluting the antibody of interest; (d) applying the eluted antibody of interest to a separation media to effect separation of more than one sample constituent wherein a separated sample constituent comprises the antibody of interest or a fragment or signature peptide thereof;
and (e) establishing the mass to charge ratio of one or more separated sample constituents by mass spectrometry, wherein the antibody of interest is selected from (a) a first anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID
NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
In some embodiments, the method further comprises treating the biological sample with a protease after incubation with the immunoaffmity beads and prior to or after eluting the antibody of interest. In some embodiments, the protease is tr3õpsin, In some embodiments of the method, the anti-idiotypic antibody is biotinylated. In some embodiments, the anti-idiotypic antibody binds to streptavidin coated paramapetic immunoaffinity beads.
In some embodiments of the method, the anti-idiotypic antibodies are bound to the immunoaffinity beads prior to contact and incubation with the biological sample.
In some embodiments of the method, the immunoaffinity bead is a magnetic bead.
In some embodiments of the method, the separation media is a chromatography support.
In various embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
In various embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2 and the anti-idiotypic antibody comprises three heavy chain hypervariable regions (IIVR-H1 IIVR-142 and HVR-113) and three light chain .hypervariable regions (IIVR-L1, HVR-L2 and HVR-L3), wherein: (a) IIVR-H1 comprises the amino acid sequence of SEQ ID NO: 13; (h) HVR-H2 comprises the amino acid sequence of SEQ
ID NO:
14; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:
18. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7.
In various embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4 and the anti-idiot:rale antibody comprises three heavy chain hypervariable regions (HVR-H1, FIVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19; (h) HVR-H2 comprises the amino acid sequence of SEQ
ID NO:
20; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO:
24. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID
NO: 9 and the light chain sequence of SEQ ID NO; 11.
.6 In some embodiments of any of the methods disclosed above, the the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I
and the light chain sequence of SEQ ID NO: 2 and the anti-idotypic antibody binds to at least one HVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain sequence of SEQ ID NO: 2.
In some embodiments of any of the methods disclosed above, the the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idotypic antibody binds to at least one HVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In some embodiments, the anti-idiotniic antibody specifically binds to HVR-H2 (SEQ ID NO: 36) of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idotypic antibody binds to an epitope on the anti-HCMV antibody that is comprised within an amino acid sequence selected from SEQ ID NO: 25 or SEQ ID NO: 26.
In some embodiments of any of the methods disclosed above, the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4 and the anti-idiotypic antibody binds to an epitope on the anti-HCMV antibody that is comprised within an amino acid amino acid sequence selected from SEQ
ID NO: 27 or SEQ ID NO: 28.
In some embodiments of any of the methods disclosed above, the antibody of interest is a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I
and the light chain sequence of SEQ ID NO: 3 and (b) a second anti -HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4.
In some embodiments of any of the methods disclosed above, the anti-idiotypic antibody binds to the antibody of interest and not to at least one other anti-HCMV
antibody in the sample.
In various embodiments of any of the methods disclosed above, the biological sample is isolated from a human subject. In some embodiments, the the human subject has been treated with an anti-HCMV antibody selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain sequence of SEQ ID
NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
'7 In some embodiments of any of the methods disclosed above, the method further comprises using a standard curve to determine the level of the antibody of interest compared to a known level.
In some embodiments of any of the methods disclosed above, the biological sample is blood, plasma or serum. In some embodiments, the sample is serum.
The invention further provides for a kit. In an embodiment, the invention provides an immunoassay kit for specifically detecting in a biological sample an antibody of interest selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ
ID NO: I and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO:
4; and (c) combination thereof, the kit comprising: (a) a container containing, as a capture reagent, an anti-idiotypic antibody that specifically binds to the antibody of interest; (1)) a container containing a detectable antibody that binds to the antibody of interest; and (c) instructions for detecting said antibody of interest. in some embodiments, the kit is useful in an ELISA
method for detecting the antibody of interest.
In some embodiments, the kit further comprises a solid support for the capture reagent. In some embodiments, the capture reagent is immobilized on the solid support. In some embodiments, the capture reagent is coated on a microliter plate.
In various embodiments, the anti-idiotypic antibody is one or more of any of the anti-antibodies disclosed above. In some embodiments, the anti-idiotypic antibody is selected from (a) a first anti-idiotypic antibody comprising the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7; (b) a second anti-idiotypic antibody comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO:
11; and (c) a combination thereof In some embodiments, the anti-idiotypic antibody specifically binds to an anti-HCMV
antibody comprising at least one heavy chain hypervariable region selected from the group consisting of NO: 13-24. hi one other embodiment, the antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-112 and HVR-H3) and three light chain hypervariable regions (HVR-L1, IIVR-L2 and IIVR-1.3), wherein: (a) HVR-Hl comprises the amino acid sequence of SEQ ID NO: 13; (b) HYR-H2 comprises the amino acid sequence of SEQ
ID NO:
14; (c) IIVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) HVR-IL1 comprises the amino acid sequence of SEQ ID NO: 16; (e) .HVR-1.2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-1,3 comprises the amino acid sequence of SEQ ID NO:
18. In another embodiment, the antibody comprises three heavy chain hypervariable regions (HVR-111, HVR-H2 and HVR-F13) and three light chain hyper-variable regions (HVR-L1, HVR-1.2 and EIVR-L3), wherein: (a) HVR-1-11 comprises the amino acid sequence of SEQ ID
NO: 19; (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22; (e) HVR-1.2 comprises the amino acid sequence of SEQ ID NO: 23;
and (0 HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
The invention also provides isolated nucleic acid encoding the anti-idiotypic HCMV
antibodies of the invention. The invention also provides host cells comprising the nucleic acid encoding such antibodies. The invention further provides a method of producing an antibody comprising culturing the host cells containing the nucleic acid encoding the antibody so that the antibody is produced. The method may further comprise recovering the antibody from the host cell.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows an amino acid sequence alignment of the heavy chain variable region (VH) of murine inAb 4.25B10,15 (SEQ ID NO: 5) with the mouse germline heavy chain variable domain region IGHV1-54*03 (SEQ ID NO: 6). The hypervariable regions (HVRs) are boxed.
Amino acid residues that differ from the mouse germline sequence are highlighted.
Figure 2 shows an amino acid sequence alignment of the light chain variable region (XL) of murine mAb 4,25B10.15 (SEQ ID NO: 7) with the mouse germline heavy chain variable domain region IGKV5-39*01 (SEQ ID NO: 8). The amino acids are numbered according to Kabat numbering. The hypervariable regions (HVRs) are boxed.
Figure 3 shows an amino acid sequence alignment of the heavy chain variable region (VH) of murine inAb 1.9E1.1 (SEQ ID NO: 9) with the mouse germline heavy chain variable domain region IGHV1-50*01 (SEQ ID NO: 10). The hypervariable regions (HVRs) are boxed.
Amino acid residues that differ from the mouse germline sequence are highlighted.
Figure 4 shows an amino acid sequence alignment of the light chain variable region (VL) of murine mAb 1.9E1,1 (SEQ ID NO: 11) with the mouse germline heavy chain variable domain region IGLVI*01 (SEQ ID NO: 12). The amino acids are numbered according to Kabat numbering. The hypervariable regions (HVRs) are boxed. Amino acid residues that differ from the mouse germline sequence are highlighted.
Figure 5 shows an anti-HCMV PK ELISA format whereby a biotin-conjugated anti-HCMV idiotypic antibody (e.g. 1.9E1 .1) binds to a streptavidin coated plate, and to a therapeutic anti-HCMV antibody in solution, The complex is then bound by a mouse anti-human IgG Fey antibody conjugated to HRP for chemiluminescent detection.
Figure 6 shows the binding activity of various purified anti-idiotypic antibodies to the anti-HCMV antibodies anti-CI and anti-gH as determined using the anti-HCMV PK
ELISA
described in Example 2.
Figure 7 shows the recovery of individual serum samples spiked with the anti-HCMV
antibody anti-gH using anti-idiotypic monoclonal antibody 1.9E1.1 as the capture antibody in the anti-HCMV PK ELISA assay described in Example 2.
Figure 8 shows an anti-HCMV PK ELISA format whereby an anti-HCMV idiotypic antibody (e.g. 4.25B10.15) is immobilized on a solid support for binding to a therapeutic anti-HCMV antibody. The complex is then bound by a mouse anti-human IgG Fey antibody conjugated to HRP for chemiluminescent detection.
Figure 9 shows the recovery of individual serum samples spiked with the anti-HCMV
antibody anti-CI using anti-idiot3pic monoclonal antibody 4.25B10.15 or 4.23F9.5 as the capture antibody in the anti-HCMV PK ELISA described in Example 3. Anti-CI was spiked at either at 4 1.1g/mL (high) or 0.5 p,g/ML (low) for the pool sample and samples 1-4; at either 2 ptglmi...
(high) or 0.6 tug/mi., (low) for sample 5, and at either 7,62 p.g/mL (high) or 0.476 (low) for samples 6 and 7.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I, DEFINITIONS
"Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a I :1 interaction between members of a binding pair (e.g., antibody and antigen).
The affinity of a molecule X fbr its partner Y can generally be represented by the dissociation constant (K(i). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
t-(y An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab`)2;
diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG,õ
IgG3, IgG4, IgA I, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, a, 7, and IA, respectively.
The term "detecting" is used in the broadest sense to include both qualitative and quantitative measurements of a target molecule. In one aspect, the detecting method as described herein is used to identify the mere presence of the antibody of interest in a biological sample. In another aspect, the method is used to test whether the antibody of interest in a sample is present at a detectable level. In yet another aspect, the method can be used to quantify the amount of the antibody of interest in a sample and further to compare the antibody levels from different samples.
The term "biological sample" refers to any biological substance that may contain an antibody of interest. A sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, itreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, and other constituents of the body that may contain the antibody of interest. In various embodiments, the sample is a body sample from any animal. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject. In some embodiments, the biological sample is from clinical patients or patients treated with a therapeutic anti-HCMV antibody or antibodies. In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient.
The term "capture reagent" or "coat antibody" refers to an anti-idiotypic antibody or mixture of such antibodies that bind an idiotype of the antibody of interest and are capable of binding and capturing the antibody of interest in a biological sample such that under suitable conditions, the complex of capture reagent and antibody of interest can be separated from the rest of the sample.
An "anti-idiotypic antibody," as used herein, is an antibody that binds to the VH andior V. domain of the cognate antibody, in this case the antibody of interest.
Typically, such anti-idiotypic antibodies are prepared by immunizing a mammal such as a mouse with the antibody of interest and producing a hybridoma and selecting from the panel of antibodies derived from the hybridoma those antibodies that give the cleanest signal in the assay, whether for the capture reagent or the detectable antibody. Typically, the capture reagent is immobilized or immobilizable. Such anti-icliotypic antibodies are monoclonal antibodies and can be for example, rodent antibodies such as murine or rat antibodies.
The terms "anti-Complex I antibody," "anti-CI antibody" or "anti-CI," as used herein, refers to an anti-HCMV antibody comprising the heavy chain variable domain of SEQ ID NO: I
and the light chain variable domain of SEQ ID NO: 2 or an antibody comprising at least one HVR region, as shown below:
EVQLVQSGAEVKKPGASVKVS CKAS GYTFTNYGMNWVRQAPGQGLEWI
GWINTYTGEPTYADDFKGRVT I TRDTS TS TAYLE LS SLRS EDTAVYYC
AR SWYYITS NYWYFDVIIGQG T LVT VS S (SEQ ID NO: 1) The bold, underlined sequences correspond to HVR-HI (SEQ ID NO: 29), HVR-H2 (SEQ ID NO: 30) and HVR-H3 (SEQ ID NO: 31) of SEQ ID NO: 1.
SVLTQS PSASAS LGASVKLTCTLS SQRSTYT I EWYQQQPGKGPRYLMK
LKKDGSHSTGDGI PDRE S GS S SGADRYLT I SNLQS EDEADYYCGVGDT
IKEQFVYVFGGGT KLTVLG (SEQ ID NO: 2) The bold, underlined sequences correspond to HVR-L1 (SEQ ID NO: 32), HVR-L2 (SEQ ID NO: 33) and HVR-L3 (SEQ ID NO: 34) of SEQ ID NO: 2.
The term "anti-gH antibody," or "anti-gH," as used herein, refers to an anti-HCMV
antibody comprising the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4 or an antibody comprising at least one HVR
region, as shown below.
EEQVLE SOGGLATKPGGSLRLS CAA'S GFTF SPYSVFWVRQAPGRGLEWV
SS INSNSRYKYYADSVKGR.FT I SRDNAENS I FLQMNSLRAEDTAVYYC
ARDRSYYAFSSGSLSDYYYGLDVWGQGTLVTVSS (SEQ ID NO: 3) The bold, underlined sequences correspond to HVR-Hl (SEQ ID NO: 35), HVR-H2 (SEQ ID NO: 36) and HVR-H3 (SEQ ID NO: 37) of SEQ ID NO: 3.
DIVMTQS PLS LSVTPGE PAS IS CRS SQSLLHTNGYNYLDWYVQKPGQS
PQLL ThASNRAS GVPDRF SGS GS GTD FMK SRVETEDVGVYYCMQA
LQIPIZTFGQGTKVEIK (SEQ ID NO: 4) The bold, underlined sequences correspond to IIVR-1.,1 (SEQ ID NO: 38), HVR-1,2 (SEQ ID NO: 39) and HVR-L3 (SEQ ID NO: 40) of SEQ ID NO: 4.
An "anti-CI idiotypic antibody," as used herein, is one that specifically binds to an anti-Cl monoclonal antibody having the heavy chain variable domain sequence of SEQ
ID NO: 1 and the light chain variable domain sequence of SEQ ID NO: 2, or to an anti-C1 antibody comprising all six hypervariable regions of an anti-CI monoclonal antibody having the heavy chain variable domain sequence of SEQ ID NO: I and the light chain variable domain sequence of SEQ ID NO:
2 (e.g., SEQ ID NOs: 29-34), with sufficient specificity and affinity to be useful in detection of anti-CI, An "anti-gH idiotypic antibody," as used herein, is one that specifically binds to an anti-gH monoclonal antibody having the heavy chain variable domain sequence of SEQ
ID NO: 3 and the light chain variable domain sequence of SEQ ID NO: 4, or to an anti-gH
antibody comprising all six hypervariable regions of an anti-gIi monoclonal antibody having the heavy chain variable domain sequence of SEQ ID NO: 3 and the light chain variable domain sequence of SEQ ID NO:
4 (e.g,, SEQ ID NOs: 35-40), with sufficient specificity and affinity to be useful in detection of anti-gH.
The term "detectable antibody" refers to an antibody that binds the antibody of interest and is capable of being detected either directly through a label amplified by a detection means, or indirectly through, e.g., another antibody that is labeled. In some embodiments, the detectable antibody is an antibody from a non-human species that binds to human antibodies. In some embodiments, the detectable antibody is an anti-idiotypic antibody or mixture of such antibodies that bind an idiotype of the antibody of interest. For direct labeling, the antibody is typically conjugated to a moiety that is detectable by some means. In some embodiments, the detectable antibody is conjugated to horseradish peroxidase.
The tenu "detection means" refers to a moiety or technique used to detect the presence of the detectable antibody through signal reporting that is then read out in the assay herein. It includes reagents that amplify the immobilized label such as the label captured onto a microliter plate.
The term "Fe region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fe regions and variant Fe regions. In one embodiment, a human IgG
heavy chain Fe region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fe region may or may not be present.
Unless otherwise specified herein, numbering of amino acid residues in the Pc region or constant region is according to the :EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR
domains: FR!, HU, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H (L1)-FR2-H2(1L2)-FR3-H3(L3)-FR4.
The terms "frill length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Pc region as defined herein.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transfbrmants" and transformed cells,"
which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VI, or VH
framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NTH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et aL supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.
A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the FIVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e,g,, a non-human antibody, refers to an antibody that has undergone humanization.
The term "hypervariable region" or "HVR," as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (LI, L2, L3). IIVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complernentarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI ), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, sf. Mot'.
Biol. 196:901-917 (1987),) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-HI, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of Ll , 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3, (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs,"
which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H I, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of Li, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3. (See Almago and Fransson, Front. Biosci.
13:1619-1633 (2008)) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al, supra.
An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
In certain embodiments, the individual or subject is a human.
An "infant" as used herein, refers to an individual or subject ranging in age from birth to not more than about one year and includes infants from 0 to about 12 months.
An "isolated" antibody is one which has been separated from a component of its natural environment, In some embodiments, an antibody is purified to greater than 95%
or 99% purity as determined by, fbr example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), 1.5.
capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase IIPLC). For review of methods for assessment of antibody purity, see, e.g., Flatrnan et al., J. Chromatogr. B
848:79-87 (2007), An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
"Isolated nucleic acid encoding an anti-idiotypic antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or frapnents thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a.
population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin such methods and other exemplary methods for making monoclonal antibodies being described herein.
A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation, "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VT), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain, The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications andior warnings concerning the use of such therapeutic products.
"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the Skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Ivlegalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code, The ALIGN-2 program should be compiled for use on a UNIX
operating system, including digital 'UNIX V4.0DõAll sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the %
amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A
that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y
is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
A "signature peptide" of an anti-HCMV antibody refers to a proteolytic peptide (e.g., a tryptic peptide) that is exclusively present in one antibody isotype. For example, an anti-CI
signature peptide may be a tryptic peptide that is exclusively present in an anti-HCMV antibody comprising the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 2. In a further example, an anti-gH signature peptide may be a tryptic peptide that is exlcusively present in an antibody an anti-HCMV antibody comprising the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ 11) NO: 4, The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervatiable regions (HVRs). (See, e.g,, Kindt et al, Kuby Immunology, 6th. ed., W.H.
Freeman and Co., page 91 (2007)) A single VII or VI, domain may be sufficient to confer antigen-binding specificity, Furthermore, antibodies that bind a particular antigen may be isolated using a VII or VI, domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains; respectively. See, e.g., Portolano et al., .1.
Inumenol.
150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."
H. COMPOSITIONS AND METHODS
In one aspect, the invention provides anti-idiotypic antibodies that specifically bind to the humanized anti-HCMV monoclonal antibodies anti-CI and anti-gH. In certain embodiments, anti-idiotypic antibodies that bind to anti-CI are provided. In certain embodiments, antibodies that bind to anti-gH are provided. Antibodies of the invention are useful, e.g., for the detection and/or quantification of anti-CI and anti-gH in biological samples, for example, in clinical samples.
Aõ Exemplary .Antit-Idiotypie Antibodies In one aspect, the invention provides isolated anti-idiotypic antibodies that bind to anti-HCMV antibodies anti-CI or anti-g-1 with sufficient specificity and affinity to be useful in detection of anti-CI and anti-gH.
In certain embodiments, an anti -idiotypic antibody binds to an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID
NO: 2. In certain embodiments, an an anti-idiotypic antibody binds to an anti-HCMV antibody comprising all six HVRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 201n certain embodiments, an anti-idiotypic antibody binds to an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In certain embodiments, an an anti-idiotypic antibody binds to an anti-HCMV antibody comprising all six HVRs from an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4.
In some embodiments, the anti-idiot:vpic antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and IIVR-H3) and three light chain hypervariable regions (HVR-1,1, HVR-12 and HVR-L3), wherein:
(a) IIVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-LI comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ. ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
In some embodiments, the anti-idiotypic antibody comprises three heavy chain hypervari able regions (HVR-H I, HVR-H2 and HVR-H3) and three light chain hypervatiable regions (HVR-L1, HVR-1,2 and IIVR-L3), wherein:
(a) HVR-Hl comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) FIVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) IIVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-1,2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 11..
In another aspect, an anti-CI idiotypic antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-CI idiotypic antibody comprising that sequence retains the ability to bind to anti-CI, In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 5. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-CI idiotypic antibody comprises the VII sequence in SEQ ID NO: 5 including post-translational modifications of that sequence. In a particular embodiment, the VII comprises one, two or three 1-1VR,s selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO: 13, (b) .HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) IIVR-H3 comprising the amino acid sequence of SEQ ID NO: 15 In another aspect, an anti-CI idiotypic antibody is provided, wherein the antibody comprises a light chain variable domain (VIõ) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.
In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e,g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-C1 idiotypic antibody comprising that sequence retains the ability to bind to anti-Ch In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 7. in certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs in the FRs).
Optionally, the anti-CI idiotypic antibody comprises the VL sequence in SEQ ID
NO: 7, including post-translational modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:
17; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18.
In another aspect, an anti-CI idiotypic antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH
and VL
sequences in SEQ ID NO: 5 and SEQ ID NO: 7, respectively, including post-translational modifications of those sequences.
In another aspect, an anti-gH idiotypic antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-gH idiotypic antibody comprising that sequence retains the ability to bind to anti-gH. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 9. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (ix., in the FRs).
Optionally, the anti-gH idiotypic antibody comprises the VH sequence in SEQ ID NO: 9 including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) IFIVR-H1 comprising the amino acid sequence of SEQ ID
NO: 19, (b) IIVR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-H3 comprising the amino acid sequence of SE() ID NO: 21.
In another aspect, an anti-gli idiotypic antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:
11. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-gH
idiotypic antibody comprising that sequence retains the ability to bind to anti-gH. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ
ID NO: 11. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the FIFV.Rs (i.e., in the FRs), Optionally, the anti-gH idiotypic antibody comprises the VL sequence in SEQ ID NO: II, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L I
comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:
24.
In another aspect, an anti-g1-1 idiotypic antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH
and VL
sequences in SEQ ID NO: 9 and SEQ ID NO: 11, respectively, including post-translational modifications of those sequences.
In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-HCMV idiotypic antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-CI idiotypic antibody comprising a VH sequence of SEQ ID NO: 5 and a VL sequence of SEQ ID NO: 7. in certain embodiments, an antibody is provided that binds to the same epitope as an anti-gH idiotypic antibody comprising a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: I. In certain.
embodiments, an antibody is provided that specifically binds to at least one HVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: I and the light chain.
sequence of SEQ ID NO: 2. In certain embodiments, an antibody is provided that specifically binds to at least one IIVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4, In certain embodiments, an antibody is provided that specifically binds to HVR-H2 (SEQ ID NO: 36) of an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID NO: 4. In certain embodiments, an antibody is provided that binds to an epitope comprised within an amino acid amino acid sequence selected from SEQ ID NO:
25, SEQ ID
NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO: 25 orSEQ ID NO: 26. In some embodiments, the anti-idiotypic antibody binds to an epitope that is comprised within an amino acid amino acid sequence selected from SEQ ID NO:
27 or SEQ ID NO: 28.
In a further aspect of the invention, an anti-HCMV idiotypic antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, an anti-HCMV idiotypic antibody is an antibody fraginent, e.g., a Fv, Fab, Fab', scFv, diabody, or P(a1:02 fragment, In another embodiment, the antibody is a full length antibody, e.g., an intact .1gG1 antibody or other antibody class or isotype as defined herein, A. Antibody Production A description follows as to exemplary techniques for the production of the anti-idiotypic antibodies used in accordance with the present invention.
1. Polvelonal Antibodies The antibodies of the invention may comprise polyclonal antibodies, Methods of preparing polycional antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include anti-CI, anti-gH, an antigen binding fragment thereof, or fusion proteins thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocrmin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation, The mammal can then be bled, and the serum assayed for anti-idiotypic antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus.
2, Monoclonal Antibodies The antibodies of the invention may alternatively be monoclonal antibodies.
Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e/g/ U.S.
Patent No.
4,816,567).
In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myelorria cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp,59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (11GPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfiised parental cells, Myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-Il mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, .S, Immunol.õ
133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp, 51-63 (Marcel Dekker, Inc., New York, 1987)), Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem,, 107:220 (1980).
Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp,59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-1`,AEM
or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.gõ by i,p. injection of the cells into mice.
The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, fur example, affinity chromatography (e.g.õ using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of =rine antibodies). The hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coil cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or rnyeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Cum Opinion in Immunol., 5:256-262 (1993) and Phickthun, Irnmunol, Revs.
130:151-188 (1992).
In a further embodiment, monoclonal antibodies or antibody fragments can be isolated.
from antibody phage libraries generated using the techniques described in McCafferty et al,, Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.
Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Biorrechnology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridorna techniques for isolation of monoclonal antibodies.
In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened for against the desired antigen. Clones expressing Fv .fraguients capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.
Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and .VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev, Immunol., 12: 433-455 (1994).
Repertoires of VH and VL genes can be separately cloned by polyrnerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev, Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., E,1\11B0 1, 12: 725-734 (1993).
Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Screening of the libraries can be accomplished by various techniques known in the art.
For example, anti-CI or anti-gH can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries.
The selection of antibodies with slow dissociation kinetics (and good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al.. Biotechnol.õ 10: 779-783 (1992).
Any of the anti-idiotypic antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-idiotypic antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD
(1991), vols. 1-3.
3. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
a) Substitution, insertion, and Deletion Variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs.
Conservative substitutions are Shown in Table 1 under the heading of "conservative substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g,, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
TABLE
Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; He Val Arg (R) Lys; Gin; Mn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Gin; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Giu Mn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; .Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Lou _________________________ Norleucine Leu (L) .Norleucine; lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin: Asn Arg Met (M) Leu; Phe; lie Len Phe (F) Try; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser TrP (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Lett .Norleueine Amino acids may be grou.ped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Len, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asti, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe, Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg% a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g, improvements) in certain biological properties (e.g., increased affinity, reduced irnmunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity), Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity.
Such alterations may be made in HVR "hotspots," i.e,, residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g,, Chowdhury, Methods Mol. Blot. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH
or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.
Such alterations may be outside of MIR "hotspots" or SDRs. In certain embodiments of the variant VH and VI., sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., al anine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal .methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
B. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-idiotypic antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light andjor heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VI.: of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In one embodiment, a method of making an anti-Complex I
antibody or anti-gH antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of an anti-Complex I antibody or an anti-gH
antibody, nucleic acid encoding an antibody, e,g., as described above, is isolated and inserted into one or more vectors for further cloning andlor expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fe effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S.
Patent Nos.
5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol.
248 (B.K,C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat, Biotech.
22:1409-1414 (2004), and Li et al., Na!, Biotech. 24:210-215 (2006).
Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera fragiperda cells.
Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos.
5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing .PLANTIBODIESTm technology for producing antibodies in trar3.sgenic plants).
Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., I. Gen Prot'. 36:59 (1977); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol, Reprod.
23:243-251 (1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76);
human cervical carcinoma cells (HELA); canine kidney cells avIDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g, in Mather et al.õ4nnals.N.Y. Acad.
Sri. 383:44-68 (1982); MRC 5 cells; and FS4 cells, Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc.
Natl. Acad, Sri.
USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B,K,C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).
C.% Assays Anti-HCMV idiotypic antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
L Binding assays and other assays In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.
In another aspect, competition assays may be used to identify an antibody that competes fur binding of an anti-hCMV antibody with anti-HCMV idiotypic antibodies described herein, In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) of anti-Cl. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) of anti-gH.
Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol, 66 (Humana Press, Totowa, NJ).
In an exemplary competition assay, immobilized anti-HCMV antibody is incubated in a solution comprising a first labeled antibody that binds to the anti-HCMV
antibody, respectively and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to anti-HCMV antibody. The second antibody may be present in a hybridoma supernatant. As a control, immobilized anti-HCMV antibody is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the .first antibody to the anti-HCMV
antibody, excess unbound antibody is removed, and the amount of label associated with immobilized anti-HCMV antibody is measured. If the amount of label associated with immobilized anti-HCMV antibody is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to anti-HCMV antibody. See Harlow and Lane (1988) Antibodies: A
Laboratory Manual ch,14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Competition assays can also be performed in a manner as described above with FACS
using cells transfected with anti-HCMV antibody and expressed on the cell surface.
Additionally, ELBA with anti-HCMV antibody can also be used in a competition assay.
ft Methods and Compositions for Detection In certain embodiments, any of the anti-idiotypic antibodies, or compositions comprising such antibodies, as provided herein, are useful 1-br detecting the presence of anti -HCMV
antibodies anti-CI or anti-gH in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample is a 'biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, and other constituents of the body that may contain the antibody of interest.
In various embodiments, the sample is a body sample from any animal. In some embodiments, the sample is from a marmnal. In some embodiments, the sample is from a human subject, for example, when measuring an antibody such as a humanized antibody in a clinical sample. In some embodiments, the biologicai sample is from clinical patients or a patient treated with a.
therapeutic anti-HCMV antibody (e.g., anti-CI and/or anti-gH). In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient.
In certain embodiments, compositions comprising labeled anti-HCMV id.iotypic antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, .32:
e.g,, through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 125/, 3H, and 1311, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodarnine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No, 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, galactosidase, glu.coamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as unease and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotiniavidin, spin labels, hacteriophage labels, stable free radicals, and the like.
ELISA
In some embodiments, the anti-HCMV idiotypic antibodies are used in an ELISA
assay.
The assay described herein is an ELIS.A that utilizes anti-HCMV idiotypic antibodies as capture reagents for an antibody of interest. In the first step of the assay the biological sample suspected of containing or containing the antibody of interest is contacted and incubated with the capture (or coat) antibodies so that the capture antibodies capture or bind to the antibody of interest so that it can be detected in a detection step. The detection step involves use of a detectable antibody, which, when contacted with any of the bound antibody of interest, binds to the antibody of interest, if present. A detection means is used to detect the label on the antibody and hence the presence or amount of antibody of interest present.
In certain embodiments, the assay utilizes the following steps.
First Step In the first step of the assay herein, the biological sample suspected of containing or containing the antibody of interest as defined herein is contacted and incubated with the immobilized capture (or coat) reagents, which are anti-idiotypic antibodies directed against the antibody of interest. In some embodiments, these antibodies are monoclonal antibodies, and may be from any species. In some embodiments, the antibodies are rodent antibodies, in further embodiments murine or rat, and in further embodiments murine antibodies.
In various embodiments, the anti-idiotypic is any anti-idiotypic antibody disclosed herein.
In certain embodiments, the anti-idiotypic antibody is an antibody comprising three heavy chain hypervariable regions (IIVR-H1, FIVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-1,1, HVR-1,2 and HVR-1,3), wherein: (a) HVR-Hl comprises the amino acid sequence of SEQ ID NO: 13; (b) HVR-H2 comprises the amino acid sequence of SEQ
ID NO:
33.
14; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) FIVR-Li comprises the amino acid sequence of SEQ ID NO: 16; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) FIVR-L3 comprises the amino acid sequence of SEQ ID NO:
18. In some embodiments, the anti-idiot3,pic antibody comprises the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7. In certain embodiments, the anti-idiotypic antibody comprises three heavy chain hypervariable regions (IIVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, ITVR-L2 and HVR-13), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19; (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-12 comprises the amino acid sequence of SEQ ID NO: 23; and (f) IIVR-L3 comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ
ID NO: 11.
Immobilization conventionally is accomplished by insolubilizing the capture reagents either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S.
Pat, No. 3,720,760) or non-covalent or covalent coupling (for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g., nitric acid and a reducing agent as described in U.S. Pat. No. 3,645,852 or in Rotmans et al.; 3. Immunol.
Methods, 57:87-98 (1983)), or afterward, e.g., by immunoprecipitation. In some embodiments, the capture antibody is conjugated to biotin and is bound to a streptavidin coated surface. In other embodiments, the capture antibody is conjugated to a protein tag, such as a His tag or GST, and is bound to a suitable surface, e.g, a nickel or copper coated surface, or a glutathione coated surface.
The solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunornetric assays, including supports in the form of, e.g., surfaces, particles, porous matrices, etc. Examples of commonly used supports include small sheets, SEPHADEX gels, polyvinyl chloride, plastic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like, including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water-insoluble matrices such as cyanogen-bromide-activated carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture-reagent immobilization. In some embodiments, the immobilized capture reagents are coated on a microtiter plate. In some embodiments, the solid phase used is a multi-well microtiter plate that can be used to analyze several samples at one time, for example, a MICROTESTTm or MAXISORPTM 96-well ELISA plate such as that sold as NUNC
MAXISORBTM or IMMULONTm.
The solid phase is coated with the capture reagents as defined above, which may be linked by a non-covalent or covalent interaction or physical linkage as desired. Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein. If covalent, the plate or other solid phase is incubated with a cross-linking agent together with the capture reagent under conditions well known in the art such as for one hour at room temperature, Commonly used cross-linking agents for attaching the capture reagents to the solid-phase substrate include, e.g., 1,1-bis(diazoacety1)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3 -dithiobis(succinimidylpropionate), and bifunctional maleimid.es such as his-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-((p-azidopheny1)-dithio)propioimidate yield photoactivatable intemiediates capable of forming cross-links in the presence of light.
If 96-well plates are utilized, they may be coated with the mixture of capture reagents typically diluted in a buffer such as 0.05 M sodium carbonate by incubation for at least about 10 hours. In sonic embodiments, incubation is at least overnight, at temperatures of about 4-20 C, or about 4-8 C, and at a pH of about 8-12, about 9-10, or about 9.6. If shorter coating times (1-2 hours) are desired, one can use 96-well plates with nitrocellulose filter bottoms (Millipore MULTISCRE.ENTm) or coat at 37 C. The plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.
The coated plates are then typically treated with a blocking agent that binds non specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate. Examples of appropriate blocking agents for this purpose include, e.g., gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk. The blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, or about 1.5 to 3 hours.
After coating and blocking, the standard (purified antibody of interest) or the biological sample to be analyzed, appropriately diluted, is added to the immobilized phase. In certain embodiments the dilution rate is about 5-1.5%, or about 10%, by volume.
Buffers that may be used for dilution for this purpose include (a) phosphate-buffered saline (PBS) containing 0.5%
BSA, 0.05% TWEEN 2OTM detergent (P20), 0.05% PROCLINTM 300 antibiotic, 5 rnM
EDTA, .35.
0,25% 3((3-cholamidopropyl)dimethylammonio)- I -propanesulphonate (CHAPS) surfactant, 0.2% beta-gamma globulin, and 0.35M NaCl; (b) PBS containing 0.5% bovine serum albumin (BSA), 0,05% P20, and 0.05% PROCLINI'm 300, pH 7; (c) PBS containing 0.5% BSA, 0,05%
P20, 0.05% PROCL1N.TM, 300, 5 inM EDTA, and 0.35 M NaC1, pH 6.35; (d) PBS
containing 0.5% BSA, 0.05% P20, 0.05% PROCLINTM 300, 5 artM EDTA, 0.2% beta-gamma globulin, and 0,35 M NaCI; and (e) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLINTM 300, 5 mM
EDTA, 0.25% CHAPS, and 0.35 M NaC1, PROCLINTM 300 acts as a preservative, and TWEEN
2OTM acts as a detergent to eliminate non-specific binding.
The amount of capture reagents employed is sufficiently large to give a good signal in comparison with the standards, but not in molar excess compared to the maximum expected level of antibody of interest in the sample. In certain embodiments, the amount of biological sample added is such that the immobilized capture reagents are in molar excess of the maximum molar concentration of free antibody of interest anticipated in the biological sample after appropriate dilution of the sample. This anticipated level depends mainly on any known correlation between the concentration levels of the free antibody of interest in the particular biological sample being analyzed with the clinical condition of the patient.
Thus, for example, an adult patient may have a maximum expected concentration of free antibody of interest in his/her serum that is quite high, whereas a child will be expected to have a lower level of free antibody of interest in his/her serum based on the doses given.
The concentration of the capture reagents may be determined by the concentration range of interest of the antibody of interest, taking any necessary dilution of the biological sample into account. The final concentration of the capture reagents may also be determined empirically to maximize the sensitivity of the assay over the range of interest. Generally, the molar excess is suitably less than about ten-fold of the maximum expected molar concentration of antibody of interest in the biological sample after any appropriate dilution of the sample.
The conditions for incubation of sample and immobilized capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation, and to ensure that any antibody of interest present in the sample binds to the immobilized capture reagent.
The incubation is accomplished at fairly constant temperatures, ranging from about 0 C to about 40 C, for example at or about room temperature. The time for incubation is generally no greater than about 10 hours, In various embodiments, the incubation time is from about 0.5 to 3 hours, or from about 1.5-3 hours at or about room temperature to maximize binding of the antibody of interest to the capture reagents. The duration of incubation may be longer if a protease inhibitor is added to prevent proteases in the biological fluid from degading the antibody of interest.
At this stage, the pH of the incubation mixture will ordinarily be in the range of about 4-9.5, or in the range of about 6-9, or about 7 to 8. The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagents to the antibody of interest being captured. Various buffers may be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, TRIS¨HCI or TRIS-phosphate, acetate, barbital, and the like, The particular buffer employed is not critical to the invention, but in individual assays one buffer may be preferred over another.
Optional Second Step In an optional second step of the assay method, the biological sample is separated (for example by washing) from the immobilized capture reagents to remove uncaptured antibody of interest. The solution used for washing is generally a buffer ("washing buffer") with a pH
determined using the considerations and buffers described above for the incubation step, with a pH range of about 6-9. The washing may be done three or more times. The temperature of washing is generally from refrigerator to moderate temperatures, with a constant temperature maintained during the assay period, typically from about 0-40 C, or about 4-30 C. For example, the wash buffer can be placed in ice at 4 C in a reservoir before the washing, and a plate washer can be utilized for this step. A cross-linking agent or other suitable agent may also be added at this stage to allow the now-bound antibody of interest to be covalently attached to the capture reagents if there is any concern that the captured antibody of interest may dissociate to some extent in the subsequent steps.
Third Step In the next step, the immobilized capture reagents with any bound antibody of interest present are contacted with detectable antibody at a temperature of about 20-40 C, or about 36-38 C, with the exact temperature and time for contacting the two being dependent primarily on the detection means employed, For example, when 4-methylumbelliferyl-P-galactoside (MUG), streptavidin-HRP, or streptavidin-P-galactosidase is used as the means for detection, the contacting may be carried out overnight (e.g,, about 15-17 hours or more) to amplify the signal to the maximum, While the detectable antibody may be a polyclonal or monoclonal antibody, preferably it is a monoclonal antibody, to reduce background noise. In some embodiments, the same anti-idiotnlic antibody is used for coat and detection in the assay. In other embodiments, different anti-idiotypic antibodies can be used for coat and detection which are selected so that the background noise is minimized.
In some embodiments, the detectable antibody is an antibody from a non-human species that binds to human antibodies, In some embodiments, the detectable antibody is an anti-huIgG
Fe antibody. In some embodiments, the detectable antibody is a mouse anti-111.4G Fry antibody.
In some embodiments, the detectable antibody is directly detectable. In certain embodiments, the detectable antibody is biotinylated. In such cases, the detection means for the biotinylated label may beavidin or streptavidin-HRP, and the readout of the detection means may be fluorimetric or colorimetric, in some embodiments, the antibody is conjugated to HRP, and the detection means is calorimetric.
A molar excess of detectable antibody with respect to the maximum concentration of free antibody of interest expected (as described above) is added to the plate after it is washed. This antibody (which is directly or indirectly detectable) is a monoclonal antibody, although any antibody can be employed. The affinity of the detectable antibody must be sufficiently high that small amounts of the free antibody of interest can be detected, but not so high that it causes the antibody of interest to be pulled from the capture reagents.
Fourth Step In the last step of the assay method, the level of any free antibody of interest from the . sample that is now bound to the capture reagents is measured using a detection means for the detectable antibody. If the biological sample is from a clinical patient, the measuring step comprises comparing the reaction that occurs as a result of the above three steps with a standard curve to determine the level of antibody of interest compared to the known amount.
The antibody added to the immobilized capture reagents will be either directly labeled, or detected indirectly by addition, after washing off of excess first antibody, of a molar excess of a second, labeled antibody directed against IgCI of the animal species of the first antibody. In the latter, indirect assay, labeled antisera against the first antibody are added to the sample so as to produce the labeled antibody in situ.
The label used for either the first or second antibody is any detectable functionality that does not interfere with the binding of free antibody of interest to the anti-idiotypic antibodies.
Examples of suitable labels are those numerous labels known for use in immunoassay, including moieties that may be detected directly, such as fluorochrome, cherniluminscent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected.
Examples of such labels include the radioisotopes 32P, 14C, 125I, 3H, and 1311, fluorophores such as rare-earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g,, firefly luciferase and bacterial luc,iferase (U.S. Pat. No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, HRP, alkaline phosphatase, galactosidase, glucoarnylase, lysozyme, saecharide oxidases, e.g,, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as unease and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin (detectable by, e.g., avidin, streptavidin, streptavidin-HRP, and streptavidin-P-galactosidase with MUG), spin labels, bacteriophage labels, stable free radicals, and the like.
Conventional methods are available to bind these labels covalently to proteins or polweptides. For instance, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat.
Nos. 3,940,475 (fluorimetry) and U.S. Pat, No. 3,645,090 (enzymes); Hunter et al,, Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Irnmunol.
Methods, 40:219-230 (1981); and Nygren, J. Histochem. and Cytochemõ 30:407-412 (1982), The conjugation of such label, including the enzymes, to the antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques.
See, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol, 73 (Academic Press, New York, New York, 1981), pp. 147-166. Suitable commercially available labeled antibodies may also be used.
Following the addition of last labeled antibody, the amount of bound antibody is determined by removing excess unbound labeled antibody through washing and then measuring the amount of the attached label using a detection method appropriate to the label, and.
correlating the measured amount with the amount of the antibody of interest in the biological sample. For example, in the case of enzymes, the amount of color developed and measured will be a direct measurement of the amount of the antibody of interest present.
Specifically, if HRP is the label, the color may be detected using the substrate TMD, using a 450 mn read wavelength and a 620 or 630 nm reference wavelength.
In one example, after an enzyme-labeled second antibody directed against the first unlabeled antibody is washed from the immobilized phase, color or chemilurniniscence is developed and measured by incubating the immobilized capture reagent with a substrate of the enzyme. Then the concentration of the antibody of interest is calculated by comparing with the color or cherniluminescence generated by the standard antibody of interest run in parallel.
2 Mass Spectrometry In some embodiments, the anti-HCMV idiotypic antibodies are used in a mass spectrometry assay for anti-HCMV antibodies anti-CI and/or anti-gH. The assays described herein utilize anti -HCMV idiotypic antibodies for immunoaffinity capture of anti-HCMV
antibodies from a biological sample. The sample may be further processed using a separation technique, such as chromatography, prior to quantification of the anti-HCMV
antibodies by mass spectroscopy. In some embodiments, characteristic peptide fragments are produced by proteolysis, and the chosen signature peptides are measured as surrogate analytes for the anti-HCMV antibodies. In certain embodiments, the surrogate peptides are quantified using HPLC
with detection by tandem mass spectroscopy (MS/MS).
Processing Biological Samples An anti-HCMV antibody selected from anti-CI, anti-gH, or a combination thereof is administered to a mammal, such as a human, or contacted with a biological source selected from a tissue, cell culture, plasma or serum. Analysis from serum and plasma samples is known to be problematic due to their high proteomic background, i.e. many proteins and other analytes. After a certain period of time, ranging from minutes, hours, days after administration, a biological sample comprising the anti-HCMV antibody, or fragment thereof is collected.
The biological sample may be collected by any means, including withdrawing a fluid by syringe or cannula. The biological sample may be blood or blood products such as serum, plasma or the like or other body fluid containing the antibody of interest.
The biological samples are processed to form analysis samples by conventional procedures including: formulating, immobilizing, centrifugation, isolating, digesting, inducing or preventing blood cell clotting, hydrolyzing, or purifying.
Processing biological samples serves to remove impurities and reduce sample heterogeneity which may hinder separation of the sample constituents, or obscure data collection or analysis. Alternatively, or in addition to, processing simplifies sample handling, preserves from degradation, minimizes sample volume, or selects for the sample constituents (analytes) of interest in the mass spectrometric analysis. Alternatively, or in addition to, processing converts biological samples into metabolites, fragments, or derivatives which are of interest in determining drug metabolism or pharmacokinetic effects.
Capturin = Processed Analysis Samples The antibody is captured on immunoaffinity beads where the beads have an immobilized anti-idiotypic antibody specific for the administered anti-HCMV antibody. In various embodiments, the anti-idiotypic is any anti-idiot5pic antibody disclosed herein. The anti-idiotypic antibody specific for the administered anti-HCMV antibody may be conjugated to the immunoaffinity beads using any suitable method known in the art. In some embodiments, the anti-idiotypic specific for the administered anti-HCMV antibody is biotinylated and bound to streptavidin coated paramagnetic beads through strong biotin-streptavidin interaction (K0-10-15 M). Rationales for using streptavidin coated paramagnetic beads include: (i) the strong streptavidin-biotin interaction (Ki)=10-15 M), (ii) the immobilized streptavidinibiotinylated analyte is a proven method, (iii) the high binding capacity (sufficient material for intact proteins), (iv) low non-specific binding, (v) elution of sample with mass spectrometry-compatible solvents, (vi) good sample recovery from beads, and (vii) ease of use and amenable fbr automation.
The immunoaffinity bead may comprise a porous polymer monolith and may be configured in a fiow-through channel in fluid communication with a collection reservoir. The beads may be contained in a flow-through vessel, such as a column or funnel wherein the sample from the biological source is introduced at one end or orifice, and a sample is eluted from another end or orifice. The immunoaffinity beads may be distributed in a plurality of flow-through vessels, each in communication with a separate collection reservoir.
The vessels and reservoirs may be configured in a 96 microtitre well format of 12x8 columns and rows, or a 384 microtitre well format of 24x16 columns and rows for purposes of automation and reproducibility of results.
Plasma or serum samples from the mammal (biological source) that received the anti-HCMV antibody are applied to the beads by manual pipetting or automated robotic dispensing.
The beads may be configured in a well or other vessel, or configured in a column, or other flow-through device where the sample is introduced at one end or orifice, and wash effluent or eluted sample is eluted from another end or orifice. Sample constituents specific for the bead bound anti-idiotypic antibody are allowed to bind. The beads are washed to rinse off non-specific proteins and other non-specific sample constituents. Bound antibodies may be deglycosylated on the beads, e.g. with PNGaseF. The bound sample constituents may be eluted into a sample plate, with segregated receiving vessels or wells. The eluted samples may then be addressed by manual pipetting or by robotic transfer and separated by reverse phase chromatography and the separated sample constituents are analyzed by mass spectrometry.
In some embodiments, the biological sample may be digested with a protease.
Characteristic peptide fragments are produced by proteolysis, and the chosen signature peptides are measured as surrogate analytes for the anti-HCMV antibodies, In an exemplary embodiment, the biological sample may be digested with trypsin digestion. For trypsin digestion, samples may be reduced with DTT, S-carboxymethylated with sodium iodoacetate, and then digested with trypsin. Digested samples may be analyzed by a separation method, for example, reverse phase HPLC, e.g. Nucleosil CI8 column; size-exclusion chromatography (SEC), e.g, TSK
3000SWxL
column; or boronate affinity chromatography using a TSK Boronate column.
41.
Separation of Sample Constituents To form the analysis sample, the biological sample may be applied to a separation media to effect separation of more than one sample constituent. Separation methods include affinity, chromatography, and electrophoresis methods. Affinity methods include affinity chromatography, adsorption, and immobilized affinity matrices. Chromatography methods include HPLC, hydrophobic interaction (HIC), anion exchange, cation exchange, reverse-phase, normal phase, ion-pair reverse-phase, thin-layer, capillary flow, and size-exclusion.
Electrophoretic methods include single dimensional, slab gel, capillary, polyacrylamide, denaturing, native, free solution, paper, 2-dimensional, isoelectric focusing, and gradient voltage.
Other separation methods include: dialysis, centrifugation, sedimentation, floatation, precipitation, immunoprecipitatiort, and gel filtration.
Separation methods may effect separation of the constituents of the biological sample by one or more physico-chemical properties including, but not limited to, elution time, hydrophobicity, hydrophilicity, migration time, rate, velocity, chromatographic retention time, solubility, molecular volume or size, net charge, charge state, ionic charge, isoelectric point, dissociation constant (pKa), antibody affinity, electrophoretic mobility, ionization potential, dipole moment, hydrogen-bonding capability, and ion mobility in gas phase.
Low rate of flow by capillary flow infusion into the mass spectrometry inlet device facilitates sensitivity of mass detection, allowing for lower concentration analytes and higher molecular weight species such as intact proteins and antibodies to be detected and characterized.
Mass Spectrometry of Separated Sample Constituents Preparation of samples for mass spectrometric analysis can be conducted generally according to known techniques. See: "Modern Protein Chemistry: Practical Aspects", Howard, G. C. and Brown, W. E., Eds. (2002) CRC Press, Boca Raton, Florida.
The methods of the invention are appropriate for the analysis of antibody mixtures derived from biological samples where different chemical constituents of the mixture are first isolated, separated, or partially separated by one or more processes including affinity or chromatography which cause the constituents to elute sequentially or in a batch wise manner, or to be directly detected by mass spectrometry. Various structural features and properties of antibodies can be elucidated from mass spectrometry analysis including:
fragmentation, deamidation, glycation, oxidation, partial sequence information, e.g. N-terminal and C-terminal, dimer and aggregation states. One or more chemical constituents in the biological sample can be characterized in a highly specific manner by measurement of its accurate mass since the administered anti-HCMV antibody is of known sequence, structure, and molecular weight.
A variety of mass spectrometry systems capable of high mass accuracy, high sensitivity, and high resolution are known in the art and can be employed in the methods of the invention.
The mass analyzers of such mass spectrometers include, but are not limited to, quadrupole (Q), time of flight (TOF)õ ion trap, magnetic sector or FT-ICR or combinations thereof. The ion source of the mass spectrometer should yield mainly sample molecular ions, or pseudo-molecular ions, and certain characterizable fragment ions. Examples of such ion sources include atmospheric pressure ionization sources, e.g. electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and Matrix Assisted Laser Desorption Ionization (MALDI).
ESI and MALDI are the two most commonly employed methods to ionize proteins for mass 1.0 spectrometric analysis. ESI and APCI are the most commonly used ion source techniques for analysis of small molecules by LC/MS (Lee, M. "LC/MS Applications in Drug Development"
(2002) J. Wiley & Sons, New York), Surface Enhanced Laser 'Desorption Ionization (SELDI) is an example of a surface-based ionization technique that allows for high-throughput mass spectrometry (U.S.
Pat. No.
6,020,208). Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface such as a "protein chip" to interact with analytes, e.g., proteins, in solution. Such surfaces selectively interact with analytes and immobilize them thereon. Thus, the analytes of the invention can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing multiple reactive moieties at different sites on a substrate surface, throughput may be increased.
In functional systems, the mass spectrometer will accurately measure the mass of a.
chemical species of interest to within 20 ppm of its exact or calculated mass, and typically within 5 ppm or less of its exact or calculated mass. Commercially available mass analyzers can sample and record the whole mass spectrum simultaneously and with a frequency that allows enough spectra to be acquired for a plurality of constituents in the mixture to ensure that the mass spectrometric signal intensity or peak area is quantitatively representative.
This will also ensure that the elution times observed for all the masses would not be modified or distorted by the mass anal :,Ter and it would help ensure that quantitative measurements are not compromised by the need to measure abundances of transient signals.
Analytical variability may be corrected for by the use of an internal standard (IS) having physicochemical properties similar to that of the target analyte. (Mesmin et al. (2011) Bioanalysis 3: 477-480). In some embodiments, where signature peptides are measured as surrogate analytes for the anti-HCMV antibodies, stable isotope labled (SUL) peptides corresponding to the signature peptides may be used as internal standards.
(Hagman et al. (2008) Anal. Chem, 80: 1290-1296; Mesmin et al. (2010) Rapid Commun. Mass Spectrom, 24: 2875-2884).
Electrospray Ionization Mass Spectrometry (ES!) Higher sensitivity is achieved at lower flow rates due to increased analyte ionization efficiency (Gale et al (1993) Rapid Commun. Mass Spectrom. 7:1017). Thus by performing electrospray injection of a sample-containing fluid at flow rates in the nanoliter per minute range provides for accurate quantitation after proper calibration, and the high sensitivity for an analyte contained within the fluid when combined with mass spectrometry. Systems and devices including a miniaturized and consolidated micro-column and micro-column array having affinity chromatographic adsorbents, which offer high selectivity and sensitivity, and accurate qualitative analysis as front ends to MS have been reported (U.S. Pat. No, 6,811,689; U.S, Pat, No.
6,020,208; U.S. Pat. No. 6,579,719).
Masses of relatively high molecular weight compounds such as antibodies can be detected at mass-to-charge ratios (miz) that are easily determined by most mass spectrometers (typical miz ranges of up to 2000 to 3000). Electrospray ionization mass spectrometry ESI-MS, in particular, is suited for charged, polar or basic compounds and for analyzing multiply charged compounds with excellent detection limits. ES1 thus allows detection and characterization of large biomolecules, such as antibodies and antibody-drug conjugates with molecular weight (MW) of 150,000 or higher. With high-mass ions, a series of multiply charged molecular ions are typically observed. The molecular weight for positive ions is determined by multiplying the measured mlz ratio with the number of charges (n) minus the mass of the cation (C+) times the number of charges (n) on that ion, The ESI method allows the presence or absence of fragmentation to be controlled by controlling the interface lens potentials. Electrospray ionization (ES!) is compatible with liquid separation methods (front end), as well as mass spectrometric detection methods (back end) (Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications", Cole, R. B., Ed. (1997) Wiley, New York.
ESI-MS data may be acquired by averaging a number of scans together and smoothing the data to provide good peak intensity and shape. For low-mass compounds, the most abundant peaks observed are often the [M+H]+ ions in the positive-ion mode and [M-I-1]-in the negative ion mode. Doubly and triply charged ions as well as dimers may also be observed. Doubly charged positive ions will be observed at a mass (MW+2C+)/2 where MW is the molecular weight and C+ is the ionizing cation, such as HI, Nat, or NH4. Except for the very low mass compounds, the detected ions will be multiply charged. Due to the soft (low ionizing potential) conditions of EST, typically only molecular ions are observed. ESI spectra may have several molecular ion peaks that differ in the mass to charge ratio due to various numbers of charges the ion possesses, A dilute solution of a sample, e.g. ADC or other biomolecule may be slowly pumped through a hypodermic needle for EST-MS analysis, The sample may be introduced via flow injection or LC/MS. Typical flow rates range from less than 1 microliter (pi) per minute up to about one milliliter (ml) per minute, ESI is particularly suited for large biological molecules that are otherwise difficult to vaporize or ionize. The needle is held at a high voltage and the strong electric field at the end of the needle charges the nebulized solution and creates charged droplets.
The charged droplets evaporate water to ultimately yield molecular ions that travel into the vacuum chamber through a small orifice. During the process of solvent evaporation, the non-covalently bound complex is transferred from solution to gas phase, (Hu et al (1994)). Gentle desolvation conditions are generally required to maintain the intact gas-phase complex. The orifice may be heated to ensure that the ions are completely desolvated, Some MS systems may employ a counter-flowed heated gas. Charged droplets are emitted from a hypodermic needle and shrink as they evaporate solvent befOre entering a vacuum chamber. Heat and gas flows may be used to aid desolvation. The amount of sample required for EST measurements may be reduced by reducing the fluid flow by use of small capillary electrospray emitter, tips, a process known as nanoelectrospray. Natioelectrospra:,,,, methods can produce a constant sivial for about 10-30 minutes for a I pi sample. The low flow has been shown to increase the ion efficiency and reduce ion suppression. Nanoelectrospray methods are frequently used for MS/MS
protein studies (Korner et al (1996) J. Am, Soc. Mass Spectrom, 7:150-156; Mann, M.
and Wilm, M.
(1996) Anal, Chem. 68:1-8.
EST of proteins produce multiply charged ions with the number of charges tending to increase as the molecular weight increases. The number of charges on a given ionic species may be determined by methods such as: (1) comparing two charge states that differ by one charge and solving simultaneous equations; (ii) looking for species that have the same charge but different adduct masses; and (iii) examining the mass-to-charge ratios for resolved isotopic clusters, The methods of EST and ESI-MS and parameters needed to conduct these methods are well known in the art. The gentleness of the electrospray ionization process allows intact antibody conjugates to be directly detected by mass spectrometry.
In one embodiment, a Q1 mass spectrum of the protein, antibody, antibody fragment or antibody-conjugates (large molecules) is run as part of the method. A suitable quality Q1 mass spectrum of a large molecule can be obtained. Since there is potential for the protein envelope to shill, all the solvents used for chromatography are made fresh and acid is added to the elution solvent to position the spectrum envelop in the observed range. For proteins of greater than 100,000 mass units, an acid such as fOrmic acid can be used at about 0.1%
(volume) in the elution solvents, for example, both solvent A (water) and B (acetonitrile). A
stronger acid can be used, such as trifluoroacetic acid (TFA), at 0,05% (volume) TFA for both A and B solvents for proteins with less than 100,000 mass units. As the amount of formic acid is decreased, the intact glycosylated antibody, trastuzumab, picks up more charge, shifting the envelope further to the left and into the observed range of raiz (1800-3000 miz). As the declustering potential (DP) voltage is increased from about 30-120V to about 70-190V, the charge on the antibody increases even further. Thus voltage applied, solvent composition, and ion pairing agents are factors to consider and adjust. The declustering potential (DP) may be increased (ramped) to acquire enough resolution to select the best charge ion range. Linearity may be obtained over a wide range of mtz. Deglycosylation of the antibody assists quantitation of intact antibody or heavy chain, fragments or ADC. G1,2,:cosylation contributes to lower ionization efficiency and thus reduced sensitivity. When quantitating antibody or antibody fragment conjugates, cleglycosylation of the antibody may reduce the heterogeneity of the mass spectrum, increase sensitivity and thus simplifying the analysis.
Deconvolution tables are used to determine the exact mass to charge ratio (iniz) for each species to quantitated. Deconvolution software applications such as Analyst.TM, QS (Applied Biosystems, Foster City, Calif) are commercially available and/or provided with mass spectrometers. Deconvolution software generally provides the user with a table of deconvoluteti masses as well as a sub-table of miz ions used to calculate these masses.
E. Kits As a matter of convenience, the assay methods of this invention can be provided in the form of a kit. Such a kit is a packaged combination including the basic elements of (a) a capture reagent comprised of an anti-idiotypic antibody against the antibody of interest;
(b) a detectable (labeled or unlabeled) antibody that binds to the antibody of interest; and (c) instructions on how to perform the assay method using these reagents.
These basic elements are defined hereinabove.
The kit may further comprise a solid support for the capture reagents, which may be provided as a separate element or on which the capture reagents are already immobilized. Hence, the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit. In some embodiments, the capture reagents are coated on a microtiter plate. The detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies raised in a different species.
Where the label is an enzyme, the kit will ordinarily include substrates and cofactors required by the enzyme; where the label is a fluorophore, a dye precursor that provides the detectable chromophore; and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to 1-IRP or 0-galactosidase with MUG.
In various embodiments, the anti-idiotypic antibody is one or more of any of the anti-idiotypic antibodies disclosed herein. In some embodiments, the the anti-idiotypic antibody is selected from (a) a first anti-idiotypic antibody comprising the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7; (h) a second anti-idiotypic antibody comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO:
11; and (c) a combination thereof The kit also typically contains the antibody of interest as a standard (e.g., purified anti-C1 and/or anti-gH), as well as other additives such as stabilizers, washing and incubation buffers;
and the like.
The components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay.
Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
The following are examples of methods and compositions of the invention, It is understood that various other embodiments may be practiced, given the general description provided above.
Example 1: Generation of Anti Anti-CI and Anti-gil Hybridotnas Five Balble mice (Charles River Laboratories International, Inc., Wilmington, MA, USA) were hyperimmunized, in each hind footpad and intraperitoneally at 3-4 day intervals, with anti-CI or anti-g11 in an adjuvant containing metabolizable squalene, Tween 80, trehalose 6,6-dimycolate and monophosphoryl lipid A (all components obtained from Sigma Aldrich, USA).
47.
After 4 to 6 boosts, serum titers were evaluated by standard enzyme-linked immunosorbant assay (ELISA) to identify mice with positive serum titers to anti-CI or anti-gH. B
cells from spleens and popliteal lymph nodes were fused with mouse rnyeloma cells (X63.Ag80653 or P3X63Ag.U1; American Type Culture Collection, Manassas, VA, USA) by electrofusion (Hybrimune-Hybridoma Production System; Harvard Apparatus, Inc,, Holliston, MA, USA).
After 10-14 days, hybridoma supernatants were harvested and screened for CDR
specific antibody production by ELISA. All specific clones were then re-screened in a preliminary HCMIter PK. ELISA, in which candidate anti-ID materials were used as reagents to coat ELISA
plates and capture anti-C1 and anti-gH, and polyclonal anti-human antibodies were used as 1.0 detection reagents. Hybridoma clones 109E1.1, 4.25B10.15 and 2.41A2.4 showed high specific binding in the preliminary HCMV PK ELISAs after the first round of single cell per well subcloning (FACSAria cell sorter; BD Biosciences, San 'Jose, CA, USA), and therefore were scaled up (Inriova 2000 Shake Flask Format; New Brunswick Scientific, Enfield, CT, USA) for antibody production. The binding pattern of clone 2.41A2.4 in the PK assay suggested that this clone binds an epitope inclusive of the lambda light chain framework.
Supernatants were purified by affinity chromatography (MabSelect SuRe; GE Healthcare Bio-Sciences, Piscataway, NJ, USA), sterile-filtered, and stored at 4 C in PBS. Isotypes of the monoclonal antibodies were determined using the Isostrip Mouse rnAb Isotyping Kit (Roche Applied Biosciences, Indianapolis, IN, USA). The isotypes were determined to be IgGl, lambda (1.9E1.1), IgGl, kappa (4.251310.15) and IgG2a, kappa (2.41A2.4).
The binding affinities of the monoclonal antibodies were determined by Biacore analysis, as shown in Table 2.
Table 2 Monoclonal Antibody Antibody Bound Binding Affinity 4.251310.15 4.7 ni'vl 2.41A2.4 17 n_M
1.9E1.1 Anti-gH 0.1 nM
The amino acid sequences of the heavy and light chain variable domains for monoclonal antibodies 4,251310,15 and 1.9E1.1 were determined, as shown in Figures 1 and 2 for 4.251310.15, and in Figures 3 and 4 fer 1,9E1.1.
The hyper-variable regions of the heavy chain of monoclonal antibody 4.25B10.15 as shown in Figure 1 are:
HVR-H1 NYLIE SEQ ID NO: 13 HVR-H2 VINPGSGGTNYNEKFEA SEQ ID NO: 14 HVR-1-13 HGSSYWYFDV SEQ ID NO: 15 The hypervariable regions of the light chain of monoclonal antibody 4.25B10.15 as shown in Figure 2 are:
HVR-L1 RASQSISDYLH SEQ ID NO: 16 YASQSIS SEQ ID NO: 17 QNGHSFPYT SEQ. ID NO: 18 The hypervariable regions of the heavy chain of monoclonal antibody 109E1.1 as shown in Figure 3 are:
HVR-H1 DYWMY SEQ ID NO: 19 FIVR-H2 AIDTSDSYTTYNQNFKG SEQ ID NO: 20 HYR-H3 SGFFLFYYPMDY SEQ ID NO: 21 The hypervariable regions of the light chain of monoclonal antibody 1.9E1.1 as shown in Figure 4 are:
IIVR-L1 RSSTGAVTTSNYAN SEQ ID NO: 22 FIVR-L2 GTVNRAP SEQ ID NO: 23 FIVR-L3 ALWYSNHIN SEQ ID NO: 24 Example 2: ELISA Assay for Detection of Anti-g11 Figure 5 shows a schematic diagram of an ELISA assay used for detection of anti-gli (referred to as the "anti-gH clinical PK ELISA"). A biotin-conjugated anti-idiotypic antibody to ,anti-gH is bound to streptavidin-coated microplates and used to detect anti-gH in the sample.
The bound anti-gfi is detected using an HRP-conjugated mouse anti-human IgCi Peg antibody, Biotin-conjugated anti-idiotypic antibody is diluted at 500 lig/MIL in assay diluent (PBS/0.5% BSA/0.05% polysorbate 20/0.35 M NaC1/0.25% CHAPS/5 mIvI EDTA/0005 ,4 ProClin 300, pH 7.4 0.1). Roche SA plates are washed three times before use with wash buffer (PBS/0.05 /0 polysorbate 20, p1-17.46) before use. 50 of biotin-conjugated anti-idiotypic antibodies (1.9E1,1) is added to streptavidin-coated plates, and the plates are incubated for 25-35 minutes at room temperature with agitation. The curve standards, assay controls or samples are diluted 1:50 in assay diluent (described above). The diluted samples, curve standards and assay controls are added to the plates and incubated for 100-130 minutes at room temperature with agitation. The plates are washed four times. 1001AI/well of IIRP- conjugated mouse anti-huigG
Fey (8 ng/m1) is added, and the plates incubated for 1hr 5 minutes at room temperature with shaking. The plates are washed four times, and100 ul/well of TMB is added. The plates are incubated for 10-15 minutes at room temperature with agitation. 100 d of 1 M
phosphoric acid is added to each well. The plates are read using a 450 nm read wavelength and a 620 or 630 nm reference wavelength.
Figure 6 shows the binding activity of various anti-idiotypic antibodies to anti-C1 and anti-gH as tested in the preliminaay anti-HCMV human PK ELISA. The assay was carried out as follows: microliter ELISA plates were coated with anti-idiotypic monoclonal antibodies at 1 lag/int in PBS overnight. Anti-C1 and anti-gli were diluted in 1% NHS and added to the wells, being subsequently detected with 50 rigirriL (in-well concentration) of HRP-Sheep anti-huIgG.
As shown in Figure 6, only one anti-idiotypic antibody, 1.9E1.1, gave an optical density (01)) in the presence of anti-gli that was greater than two-fold the OD in the absence of anti-gH.
The other anti-idiotypic antibodies gave high background OD, suggesting cross-reaction of those anti-idiotypic antibodies with endogenous IgG in human serum (NHS).
The anti-gH clinical PK ELBA was used to quantify the amount of anti-gH spiked into sera from individual donors. Anti-gH was spiked at either high (2 lag/111W or low (0.3 p.g/mL) concentrations in neat serum. Figure 7 shows the percent recovery of individual serum spikes with anti-gli using 1.9E1.1 as the capture antibody. The assay Showed that anti-idiotypic antibody 1.9E1.1 provided good accuracy with all individual sera, spiked at both low and high anti-gH concentrations.
Example 3: ELBA Assay for Detection of Anti-CT
Figure 8 is a schematic diagram of an ELISA assay used for detection of anti-CI (referred to as the "anti-C1 clinical PK ELISA"). Anti-idiotypic antibody to anti-CI is bound to microplates and used to detect anti-CI in the sample. The bound anti-CI is detected using an :IMP-conjugated mouse anti-human IgG Fcg antibody, Microtiter plates are coated overnight at 2-8 C with 100 gl per well of 0.75 anti-C1 idiotypic antibody (in 0.05 M sodium carbonate buffer, pH9.6 0,1). The plates are washed three times with 400 l per well per cycle of wash buffer. 200 ul per well of blocking buffer (PBS/0.5% BSA/0M5% polysorbate 20/0.05% ProClin 300, pH 7,4 0.1) is added, and the plates are incubated at room temperature with shaking for 1-3 hours. Standard curves are prepared with standard/sample diluent (assay diluent with 0.5% normal pooled human serum;
assay diluent is composed of PBS/0.5 //0 BSA/0.05% polysorbate-20/0.35 M NaCl/0.25%
CHAPS/5inlvi EDTA/0.05% ProClin 300, pH 7.4 0,1). The plates are washed three times with 400 IA per well per cycle of wash buffer. Diluted standards, controls and samples are added to plates at 100 per well, in duplicate. The plates are incubated at room temperature with shaking for 2 hours 10 minutes. The plates are washed four times with 400 d per well per cycle of wash buffer. HRP
conjugates diluted in conjugate buffer (PBS10.54),43BSA/0.05% polysorbate 20/0.05% ProClin 300, pH 7.4 0,1) are added at 100 pi per well, The plates are incubated at room temperature with shaking for 1 hour 5 minutes, following which the plates are washed four times with 400 ul per well per cycle of wash buffer, 1001..d per well of TMB substrate is added, and the plates are incubated for approximately 15 minutes at room temperature with shaking. 100 ul of 1 M
phosphoric acid is added to each well. The plates are read using a 450 nni read wavelength and a 620 or 630 nm reference wavelength.
The anti-C1 clinical PK ELISA assay was carried out using plates coated with varying concentrations of capture antibody 4.25B10.15 in order to optimize the coating concentration. As shown in Table 3, plates coated with 0,75 mg/m1 of antibody 4.25B10.15 gave the optimum signal to background ratio, Background (bkgd) equals the assay signal obtained with unspiked human serum.
Table 3 Plate coat with 4.2510.15 (73427-34) coat Signalibkgd coat Signalibkgd coat Signalibkgd ' 8G8 (/m L) Iggimi. ratio 0.75tterral ratio a 5p,gimi. ratio 16 1.876 13.8 1.593 19.1 1.281 14.7 8 1.771 13.0 1.378 16.5 1.075 12.3 4 1,371 10,0 1,107 13.3 0.901 10.4 1 0.599 4.4 0.439 5,3 0.379 4,3 0.5 0.372 2.7 0.271 3.2 0,231 2.7 0.25 0.254 1,9 0.174 2.1 0.161 1.8 0.125 0.194 1.4 0.128 1.5 0.120 1.4 bkgd 0,136 0.084 0.087 The anti-CI clinical PK ELISA was run with a standard curve using anti-CI at varying concentrations spiked into 0.5% human serum pool. Results are summarized in Table 4. The assay showed a consistent dose response, and increasingly higher signal to background (SIB) ratio with increasing concentrations of anti-CI.
Table 4 Anti-Clugiml.. OD SiB ratio 20 1,970 22.3 10 1.725 19,6 5 1,203 13.6 2.5 0.743 8.4 1.25 0.431 4.9 0.625 0,261 3,0 0.313 0.174 2.0 0.156 0.134 1.5 bkgd 0.088 The anti-CI clinical PK ELISA was used to quantify the amount of anti-CI
spiked into sera from individual donors, using anti-idiotypic antibody 4.251310.15 or 4.2$F9.5 as the capture antibody. Anti-CI was spiked at either at 4 (high) or 0.5 (low) for the pool sample and samples 1-4; at either 12 p.whiL (high) or 0.6 pg/mL (low) for sample 5, and at either 7.62 li.g/mt (high) or 0,476 ggirn1_, (low) for samples 6 and 7. As Shown in Figure 9, aunti-idiotypic antibody 4.251310.15 performed better than 4,23F9,5, providing accurate spike recovery for all samples, at both high and low anti-CI concentrations.
Example 4: LC-MSINIS Assay for Quantification of Anti-CI and Anti-g11 in Human Serum An LC-MS/MS Assay was developed for quantification of anti-C1 and anti-gH in human serum. The assay used irnmunoaffinity capture to isolate two monoclonal antibodies, anti-CI and anti-gH, from human serum. Characteristic peptide fragments were then produced by proteolysis of the bound antibodies with trwsin, and the chosen signature peptides, along with their stable isotope-labeled internal standards (SIL ISs) were measured as surrogate analytes for anti-CI and anti-gH using HPLC with MS/MS detection. The signature tryptic peptides for anti-CI and anti-gH were as shown:
Anti-CI (P2) EQFVYVFOGGTK (SEQ ID NO: 25) Anti-CI (P4) DTSTSTAYLELSSLR (SEQ ID NO: 26) Anti-gH (P2) GLEWVSSINSNSR (SEQ ID NO: 27) Anti-gH (P5) LSC*AASOFTFSPYSVFWVR (SEQ ID NO: 28) Alkylated cysteine residue The SIL ISs comprised stable isotope-labeled amino acids at the amino acid positions shown in bold:
75 Anti-CI (P2) EQFVYVFGGGTK (SEQ ID NO: 25) Anti-CI (P4) DTSTSTAYLELSSLR (SEQ ID NO: 26) Anti-gH (P2) GLEWVSSINSNSR (SEQ ID NO: 27) Anti-gH (P5) LSC*AASGFTFSPYSVFWVR (SEQ ID NO: 28) Alkylated cysteine residue Signature peptides Anti-CI (P2) and Anti-gH (P2) are the primary surrogate peptides used for quantification of anti-CI and anti-gH, respectively, while Anti-CI (P4) and Anti-gH (P5) were secondary surrogate peptides used for quality control purposes. The method is applicable to the quantitation of anti-CI and anti-gH within a nominal range of 0,100 to 20,0 pg/mL, A 25-4 human serum aliquot is sufficient for analysis. Samples are stored in polypropylene tubes and kept frozen at approximately -70 'V prior to analysis.
The following sample types were extracted for evaluation purposes. Except where noted, the anti-idiotypic monoclonal antibody (anti-ID mAb) loads were 3.0 pig each per sample:
Pooled blank (MB) human serum.
Calibration Standards (CALs) fortified with anti-CI and anti-gli (combined) at the following concentrations .for each antibody: 0.100, 0.200, 0A00, LOO, 4.00, 8.00, 10,0 and 20.0 pig/mL (n=2 each level).
Quality Controls (QCs) fortified with anti-CI and anti-gH (combined) at the following concentrations for each antibody: 0.250, 2.00, 7.50 ug/m1., (n=4 each level).
Carryover blanks (CB) following high calibrators.
Over the curve dilution QC (DIL QC) prepared at anti-CI and anti-gH
concentration of 40 pg/mL and diluted 10-fold prior to aliquoting.
Specificity Samples (SP) from six individual human donors (n=1 each).
Fortified specificity samples (SPIE's) from six individual human donors fortified with anti-CI and anti-gH at a concentration of 0,200 pg/mL.
Additional Calibration Standards (CALs) fortified with anti-CI and anti-gH
(combined) at the following concentrations: 0.100, 0.200, 0.400, 1,00, 4,00, 8.00, 10.0 and 20.01.,ig/mL (n=2 each level) and containing anti-C1 anti-1D mAb and anti-gH anti-ID mAb loads of 1.50 jig each per sample.
The samples for analysis were processed by the following steps:
1. Precondition an Lmmulon 1B 96-well, flat-bottom inic,rotiter plate (Thermo, Product No. 3355) by adding 275 ul of plate-conditioning buffer (0.1:500:3.0:0,2:91.7:0.1 Tween 20 / Trizma HCI (1 M) NaCI (5 M) / ED'I'A (0.5 M)/ water / bovine serum albumin, v/v/v/v/w. Gently vortex the plate for approximately 1 min, Discard the conditioning buffer by inverting the plate, and tap plate dry on an absorbent pad.
2= A 25 jiL aliquot of serum sample is added to a mixture of 3.0 jig or 1.5 jig anti-1D mAb, 3.0 jig or 1.5 pig anti-gH anti-1D mAb and 10 mM FIBS-EP buffer (GE Healthcare, Product No, 94318) to yield a total volume of 1501,tL in the preconditioned 96-well micro-well plate.
3. Cover the plate with an adhesive film, and incubate at room temperature (RT) for 2 'vs under constant gentle shaking on a titer plate shaker.
4. The required volume of streptavidin Dynabeads M-280 (Life Technologies, Product No, 602-10) (based on 100 pit per well) is buffer exchanged with an equal volume of 10 mM HBS-EP by mixing at RT for 1-2 min. This procedure is repeated thrice.
After discarding the supernatant, the volume is made up with 10 mM HBS-EP to 50 pt so that the beads are concentrated 2-fold compared to the initial starting volume.
5. 50 III., of the concentrated beads are then added to each well from step 3 and allowed to bind at RT for 2 hrs under constant gentle shaking.
6.
Using an external magnet (Biotek 96-well Flat Magnet) and a Biotek plate washer, separate the magnetic beads and discard the unbound proteins in the supernatant. Wash the beads three times with 75 of bead washing buffer (5.0:3.0:0.2:91,8 Tiizman HC1 (1 M) /
NaCl (5 M) EDTA (0.5 M) / water, v/v/v/v) using the plate washer. Discard the supernatant after the beads are separated using the external magnet. Using the titer plate shaker, shake the titer plate between each of the three washes to resuspend the beads.
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg% a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g, improvements) in certain biological properties (e.g., increased affinity, reduced irnmunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity), Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity.
Such alterations may be made in HVR "hotspots," i.e,, residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g,, Chowdhury, Methods Mol. Blot. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH
or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.
Such alterations may be outside of MIR "hotspots" or SDRs. In certain embodiments of the variant VH and VI., sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., al anine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal .methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
B. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-idiotypic antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light andjor heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VI.: of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In one embodiment, a method of making an anti-Complex I
antibody or anti-gH antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of an anti-Complex I antibody or an anti-gH
antibody, nucleic acid encoding an antibody, e,g., as described above, is isolated and inserted into one or more vectors for further cloning andlor expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fe effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S.
Patent Nos.
5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol.
248 (B.K,C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat, Biotech.
22:1409-1414 (2004), and Li et al., Na!, Biotech. 24:210-215 (2006).
Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera fragiperda cells.
Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos.
5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing .PLANTIBODIESTm technology for producing antibodies in trar3.sgenic plants).
Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., I. Gen Prot'. 36:59 (1977); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol, Reprod.
23:243-251 (1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76);
human cervical carcinoma cells (HELA); canine kidney cells avIDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g, in Mather et al.õ4nnals.N.Y. Acad.
Sri. 383:44-68 (1982); MRC 5 cells; and FS4 cells, Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc.
Natl. Acad, Sri.
USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B,K,C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).
C.% Assays Anti-HCMV idiotypic antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
L Binding assays and other assays In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.
In another aspect, competition assays may be used to identify an antibody that competes fur binding of an anti-hCMV antibody with anti-HCMV idiotypic antibodies described herein, In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) of anti-Cl. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) of anti-gH.
Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol, 66 (Humana Press, Totowa, NJ).
In an exemplary competition assay, immobilized anti-HCMV antibody is incubated in a solution comprising a first labeled antibody that binds to the anti-HCMV
antibody, respectively and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to anti-HCMV antibody. The second antibody may be present in a hybridoma supernatant. As a control, immobilized anti-HCMV antibody is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the .first antibody to the anti-HCMV
antibody, excess unbound antibody is removed, and the amount of label associated with immobilized anti-HCMV antibody is measured. If the amount of label associated with immobilized anti-HCMV antibody is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to anti-HCMV antibody. See Harlow and Lane (1988) Antibodies: A
Laboratory Manual ch,14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Competition assays can also be performed in a manner as described above with FACS
using cells transfected with anti-HCMV antibody and expressed on the cell surface.
Additionally, ELBA with anti-HCMV antibody can also be used in a competition assay.
ft Methods and Compositions for Detection In certain embodiments, any of the anti-idiotypic antibodies, or compositions comprising such antibodies, as provided herein, are useful 1-br detecting the presence of anti -HCMV
antibodies anti-CI or anti-gH in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample is a 'biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, and other constituents of the body that may contain the antibody of interest.
In various embodiments, the sample is a body sample from any animal. In some embodiments, the sample is from a marmnal. In some embodiments, the sample is from a human subject, for example, when measuring an antibody such as a humanized antibody in a clinical sample. In some embodiments, the biologicai sample is from clinical patients or a patient treated with a.
therapeutic anti-HCMV antibody (e.g., anti-CI and/or anti-gH). In certain embodiments, the biological sample is serum or plasma. In certain embodiments, the biological sample is serum from a clinical patient.
In certain embodiments, compositions comprising labeled anti-HCMV id.iotypic antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, .32:
e.g,, through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 125/, 3H, and 1311, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodarnine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No, 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, galactosidase, glu.coamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as unease and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotiniavidin, spin labels, hacteriophage labels, stable free radicals, and the like.
ELISA
In some embodiments, the anti-HCMV idiotypic antibodies are used in an ELISA
assay.
The assay described herein is an ELIS.A that utilizes anti-HCMV idiotypic antibodies as capture reagents for an antibody of interest. In the first step of the assay the biological sample suspected of containing or containing the antibody of interest is contacted and incubated with the capture (or coat) antibodies so that the capture antibodies capture or bind to the antibody of interest so that it can be detected in a detection step. The detection step involves use of a detectable antibody, which, when contacted with any of the bound antibody of interest, binds to the antibody of interest, if present. A detection means is used to detect the label on the antibody and hence the presence or amount of antibody of interest present.
In certain embodiments, the assay utilizes the following steps.
First Step In the first step of the assay herein, the biological sample suspected of containing or containing the antibody of interest as defined herein is contacted and incubated with the immobilized capture (or coat) reagents, which are anti-idiotypic antibodies directed against the antibody of interest. In some embodiments, these antibodies are monoclonal antibodies, and may be from any species. In some embodiments, the antibodies are rodent antibodies, in further embodiments murine or rat, and in further embodiments murine antibodies.
In various embodiments, the anti-idiotypic is any anti-idiotypic antibody disclosed herein.
In certain embodiments, the anti-idiotypic antibody is an antibody comprising three heavy chain hypervariable regions (IIVR-H1, FIVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-1,1, HVR-1,2 and HVR-1,3), wherein: (a) HVR-Hl comprises the amino acid sequence of SEQ ID NO: 13; (b) HVR-H2 comprises the amino acid sequence of SEQ
ID NO:
33.
14; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15; (d) FIVR-Li comprises the amino acid sequence of SEQ ID NO: 16; (e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) FIVR-L3 comprises the amino acid sequence of SEQ ID NO:
18. In some embodiments, the anti-idiot3,pic antibody comprises the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7. In certain embodiments, the anti-idiotypic antibody comprises three heavy chain hypervariable regions (IIVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, ITVR-L2 and HVR-13), wherein: (a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19; (b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20; (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-12 comprises the amino acid sequence of SEQ ID NO: 23; and (f) IIVR-L3 comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ
ID NO: 11.
Immobilization conventionally is accomplished by insolubilizing the capture reagents either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S.
Pat, No. 3,720,760) or non-covalent or covalent coupling (for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g., nitric acid and a reducing agent as described in U.S. Pat. No. 3,645,852 or in Rotmans et al.; 3. Immunol.
Methods, 57:87-98 (1983)), or afterward, e.g., by immunoprecipitation. In some embodiments, the capture antibody is conjugated to biotin and is bound to a streptavidin coated surface. In other embodiments, the capture antibody is conjugated to a protein tag, such as a His tag or GST, and is bound to a suitable surface, e.g, a nickel or copper coated surface, or a glutathione coated surface.
The solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunornetric assays, including supports in the form of, e.g., surfaces, particles, porous matrices, etc. Examples of commonly used supports include small sheets, SEPHADEX gels, polyvinyl chloride, plastic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like, including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water-insoluble matrices such as cyanogen-bromide-activated carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture-reagent immobilization. In some embodiments, the immobilized capture reagents are coated on a microtiter plate. In some embodiments, the solid phase used is a multi-well microtiter plate that can be used to analyze several samples at one time, for example, a MICROTESTTm or MAXISORPTM 96-well ELISA plate such as that sold as NUNC
MAXISORBTM or IMMULONTm.
The solid phase is coated with the capture reagents as defined above, which may be linked by a non-covalent or covalent interaction or physical linkage as desired. Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein. If covalent, the plate or other solid phase is incubated with a cross-linking agent together with the capture reagent under conditions well known in the art such as for one hour at room temperature, Commonly used cross-linking agents for attaching the capture reagents to the solid-phase substrate include, e.g., 1,1-bis(diazoacety1)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3 -dithiobis(succinimidylpropionate), and bifunctional maleimid.es such as his-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-((p-azidopheny1)-dithio)propioimidate yield photoactivatable intemiediates capable of forming cross-links in the presence of light.
If 96-well plates are utilized, they may be coated with the mixture of capture reagents typically diluted in a buffer such as 0.05 M sodium carbonate by incubation for at least about 10 hours. In sonic embodiments, incubation is at least overnight, at temperatures of about 4-20 C, or about 4-8 C, and at a pH of about 8-12, about 9-10, or about 9.6. If shorter coating times (1-2 hours) are desired, one can use 96-well plates with nitrocellulose filter bottoms (Millipore MULTISCRE.ENTm) or coat at 37 C. The plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.
The coated plates are then typically treated with a blocking agent that binds non specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate. Examples of appropriate blocking agents for this purpose include, e.g., gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk. The blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, or about 1.5 to 3 hours.
After coating and blocking, the standard (purified antibody of interest) or the biological sample to be analyzed, appropriately diluted, is added to the immobilized phase. In certain embodiments the dilution rate is about 5-1.5%, or about 10%, by volume.
Buffers that may be used for dilution for this purpose include (a) phosphate-buffered saline (PBS) containing 0.5%
BSA, 0.05% TWEEN 2OTM detergent (P20), 0.05% PROCLINTM 300 antibiotic, 5 rnM
EDTA, .35.
0,25% 3((3-cholamidopropyl)dimethylammonio)- I -propanesulphonate (CHAPS) surfactant, 0.2% beta-gamma globulin, and 0.35M NaCl; (b) PBS containing 0.5% bovine serum albumin (BSA), 0,05% P20, and 0.05% PROCLINI'm 300, pH 7; (c) PBS containing 0.5% BSA, 0,05%
P20, 0.05% PROCL1N.TM, 300, 5 inM EDTA, and 0.35 M NaC1, pH 6.35; (d) PBS
containing 0.5% BSA, 0.05% P20, 0.05% PROCLINTM 300, 5 artM EDTA, 0.2% beta-gamma globulin, and 0,35 M NaCI; and (e) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLINTM 300, 5 mM
EDTA, 0.25% CHAPS, and 0.35 M NaC1, PROCLINTM 300 acts as a preservative, and TWEEN
2OTM acts as a detergent to eliminate non-specific binding.
The amount of capture reagents employed is sufficiently large to give a good signal in comparison with the standards, but not in molar excess compared to the maximum expected level of antibody of interest in the sample. In certain embodiments, the amount of biological sample added is such that the immobilized capture reagents are in molar excess of the maximum molar concentration of free antibody of interest anticipated in the biological sample after appropriate dilution of the sample. This anticipated level depends mainly on any known correlation between the concentration levels of the free antibody of interest in the particular biological sample being analyzed with the clinical condition of the patient.
Thus, for example, an adult patient may have a maximum expected concentration of free antibody of interest in his/her serum that is quite high, whereas a child will be expected to have a lower level of free antibody of interest in his/her serum based on the doses given.
The concentration of the capture reagents may be determined by the concentration range of interest of the antibody of interest, taking any necessary dilution of the biological sample into account. The final concentration of the capture reagents may also be determined empirically to maximize the sensitivity of the assay over the range of interest. Generally, the molar excess is suitably less than about ten-fold of the maximum expected molar concentration of antibody of interest in the biological sample after any appropriate dilution of the sample.
The conditions for incubation of sample and immobilized capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation, and to ensure that any antibody of interest present in the sample binds to the immobilized capture reagent.
The incubation is accomplished at fairly constant temperatures, ranging from about 0 C to about 40 C, for example at or about room temperature. The time for incubation is generally no greater than about 10 hours, In various embodiments, the incubation time is from about 0.5 to 3 hours, or from about 1.5-3 hours at or about room temperature to maximize binding of the antibody of interest to the capture reagents. The duration of incubation may be longer if a protease inhibitor is added to prevent proteases in the biological fluid from degading the antibody of interest.
At this stage, the pH of the incubation mixture will ordinarily be in the range of about 4-9.5, or in the range of about 6-9, or about 7 to 8. The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagents to the antibody of interest being captured. Various buffers may be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, TRIS¨HCI or TRIS-phosphate, acetate, barbital, and the like, The particular buffer employed is not critical to the invention, but in individual assays one buffer may be preferred over another.
Optional Second Step In an optional second step of the assay method, the biological sample is separated (for example by washing) from the immobilized capture reagents to remove uncaptured antibody of interest. The solution used for washing is generally a buffer ("washing buffer") with a pH
determined using the considerations and buffers described above for the incubation step, with a pH range of about 6-9. The washing may be done three or more times. The temperature of washing is generally from refrigerator to moderate temperatures, with a constant temperature maintained during the assay period, typically from about 0-40 C, or about 4-30 C. For example, the wash buffer can be placed in ice at 4 C in a reservoir before the washing, and a plate washer can be utilized for this step. A cross-linking agent or other suitable agent may also be added at this stage to allow the now-bound antibody of interest to be covalently attached to the capture reagents if there is any concern that the captured antibody of interest may dissociate to some extent in the subsequent steps.
Third Step In the next step, the immobilized capture reagents with any bound antibody of interest present are contacted with detectable antibody at a temperature of about 20-40 C, or about 36-38 C, with the exact temperature and time for contacting the two being dependent primarily on the detection means employed, For example, when 4-methylumbelliferyl-P-galactoside (MUG), streptavidin-HRP, or streptavidin-P-galactosidase is used as the means for detection, the contacting may be carried out overnight (e.g,, about 15-17 hours or more) to amplify the signal to the maximum, While the detectable antibody may be a polyclonal or monoclonal antibody, preferably it is a monoclonal antibody, to reduce background noise. In some embodiments, the same anti-idiotnlic antibody is used for coat and detection in the assay. In other embodiments, different anti-idiotypic antibodies can be used for coat and detection which are selected so that the background noise is minimized.
In some embodiments, the detectable antibody is an antibody from a non-human species that binds to human antibodies, In some embodiments, the detectable antibody is an anti-huIgG
Fe antibody. In some embodiments, the detectable antibody is a mouse anti-111.4G Fry antibody.
In some embodiments, the detectable antibody is directly detectable. In certain embodiments, the detectable antibody is biotinylated. In such cases, the detection means for the biotinylated label may beavidin or streptavidin-HRP, and the readout of the detection means may be fluorimetric or colorimetric, in some embodiments, the antibody is conjugated to HRP, and the detection means is calorimetric.
A molar excess of detectable antibody with respect to the maximum concentration of free antibody of interest expected (as described above) is added to the plate after it is washed. This antibody (which is directly or indirectly detectable) is a monoclonal antibody, although any antibody can be employed. The affinity of the detectable antibody must be sufficiently high that small amounts of the free antibody of interest can be detected, but not so high that it causes the antibody of interest to be pulled from the capture reagents.
Fourth Step In the last step of the assay method, the level of any free antibody of interest from the . sample that is now bound to the capture reagents is measured using a detection means for the detectable antibody. If the biological sample is from a clinical patient, the measuring step comprises comparing the reaction that occurs as a result of the above three steps with a standard curve to determine the level of antibody of interest compared to the known amount.
The antibody added to the immobilized capture reagents will be either directly labeled, or detected indirectly by addition, after washing off of excess first antibody, of a molar excess of a second, labeled antibody directed against IgCI of the animal species of the first antibody. In the latter, indirect assay, labeled antisera against the first antibody are added to the sample so as to produce the labeled antibody in situ.
The label used for either the first or second antibody is any detectable functionality that does not interfere with the binding of free antibody of interest to the anti-idiotypic antibodies.
Examples of suitable labels are those numerous labels known for use in immunoassay, including moieties that may be detected directly, such as fluorochrome, cherniluminscent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected.
Examples of such labels include the radioisotopes 32P, 14C, 125I, 3H, and 1311, fluorophores such as rare-earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g,, firefly luciferase and bacterial luc,iferase (U.S. Pat. No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, HRP, alkaline phosphatase, galactosidase, glucoarnylase, lysozyme, saecharide oxidases, e.g,, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as unease and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin (detectable by, e.g., avidin, streptavidin, streptavidin-HRP, and streptavidin-P-galactosidase with MUG), spin labels, bacteriophage labels, stable free radicals, and the like.
Conventional methods are available to bind these labels covalently to proteins or polweptides. For instance, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat.
Nos. 3,940,475 (fluorimetry) and U.S. Pat, No. 3,645,090 (enzymes); Hunter et al,, Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Irnmunol.
Methods, 40:219-230 (1981); and Nygren, J. Histochem. and Cytochemõ 30:407-412 (1982), The conjugation of such label, including the enzymes, to the antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques.
See, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol, 73 (Academic Press, New York, New York, 1981), pp. 147-166. Suitable commercially available labeled antibodies may also be used.
Following the addition of last labeled antibody, the amount of bound antibody is determined by removing excess unbound labeled antibody through washing and then measuring the amount of the attached label using a detection method appropriate to the label, and.
correlating the measured amount with the amount of the antibody of interest in the biological sample. For example, in the case of enzymes, the amount of color developed and measured will be a direct measurement of the amount of the antibody of interest present.
Specifically, if HRP is the label, the color may be detected using the substrate TMD, using a 450 mn read wavelength and a 620 or 630 nm reference wavelength.
In one example, after an enzyme-labeled second antibody directed against the first unlabeled antibody is washed from the immobilized phase, color or chemilurniniscence is developed and measured by incubating the immobilized capture reagent with a substrate of the enzyme. Then the concentration of the antibody of interest is calculated by comparing with the color or cherniluminescence generated by the standard antibody of interest run in parallel.
2 Mass Spectrometry In some embodiments, the anti-HCMV idiotypic antibodies are used in a mass spectrometry assay for anti-HCMV antibodies anti-CI and/or anti-gH. The assays described herein utilize anti -HCMV idiotypic antibodies for immunoaffinity capture of anti-HCMV
antibodies from a biological sample. The sample may be further processed using a separation technique, such as chromatography, prior to quantification of the anti-HCMV
antibodies by mass spectroscopy. In some embodiments, characteristic peptide fragments are produced by proteolysis, and the chosen signature peptides are measured as surrogate analytes for the anti-HCMV antibodies. In certain embodiments, the surrogate peptides are quantified using HPLC
with detection by tandem mass spectroscopy (MS/MS).
Processing Biological Samples An anti-HCMV antibody selected from anti-CI, anti-gH, or a combination thereof is administered to a mammal, such as a human, or contacted with a biological source selected from a tissue, cell culture, plasma or serum. Analysis from serum and plasma samples is known to be problematic due to their high proteomic background, i.e. many proteins and other analytes. After a certain period of time, ranging from minutes, hours, days after administration, a biological sample comprising the anti-HCMV antibody, or fragment thereof is collected.
The biological sample may be collected by any means, including withdrawing a fluid by syringe or cannula. The biological sample may be blood or blood products such as serum, plasma or the like or other body fluid containing the antibody of interest.
The biological samples are processed to form analysis samples by conventional procedures including: formulating, immobilizing, centrifugation, isolating, digesting, inducing or preventing blood cell clotting, hydrolyzing, or purifying.
Processing biological samples serves to remove impurities and reduce sample heterogeneity which may hinder separation of the sample constituents, or obscure data collection or analysis. Alternatively, or in addition to, processing simplifies sample handling, preserves from degradation, minimizes sample volume, or selects for the sample constituents (analytes) of interest in the mass spectrometric analysis. Alternatively, or in addition to, processing converts biological samples into metabolites, fragments, or derivatives which are of interest in determining drug metabolism or pharmacokinetic effects.
Capturin = Processed Analysis Samples The antibody is captured on immunoaffinity beads where the beads have an immobilized anti-idiotypic antibody specific for the administered anti-HCMV antibody. In various embodiments, the anti-idiotypic is any anti-idiot5pic antibody disclosed herein. The anti-idiotypic antibody specific for the administered anti-HCMV antibody may be conjugated to the immunoaffinity beads using any suitable method known in the art. In some embodiments, the anti-idiotypic specific for the administered anti-HCMV antibody is biotinylated and bound to streptavidin coated paramagnetic beads through strong biotin-streptavidin interaction (K0-10-15 M). Rationales for using streptavidin coated paramagnetic beads include: (i) the strong streptavidin-biotin interaction (Ki)=10-15 M), (ii) the immobilized streptavidinibiotinylated analyte is a proven method, (iii) the high binding capacity (sufficient material for intact proteins), (iv) low non-specific binding, (v) elution of sample with mass spectrometry-compatible solvents, (vi) good sample recovery from beads, and (vii) ease of use and amenable fbr automation.
The immunoaffinity bead may comprise a porous polymer monolith and may be configured in a fiow-through channel in fluid communication with a collection reservoir. The beads may be contained in a flow-through vessel, such as a column or funnel wherein the sample from the biological source is introduced at one end or orifice, and a sample is eluted from another end or orifice. The immunoaffinity beads may be distributed in a plurality of flow-through vessels, each in communication with a separate collection reservoir.
The vessels and reservoirs may be configured in a 96 microtitre well format of 12x8 columns and rows, or a 384 microtitre well format of 24x16 columns and rows for purposes of automation and reproducibility of results.
Plasma or serum samples from the mammal (biological source) that received the anti-HCMV antibody are applied to the beads by manual pipetting or automated robotic dispensing.
The beads may be configured in a well or other vessel, or configured in a column, or other flow-through device where the sample is introduced at one end or orifice, and wash effluent or eluted sample is eluted from another end or orifice. Sample constituents specific for the bead bound anti-idiotypic antibody are allowed to bind. The beads are washed to rinse off non-specific proteins and other non-specific sample constituents. Bound antibodies may be deglycosylated on the beads, e.g. with PNGaseF. The bound sample constituents may be eluted into a sample plate, with segregated receiving vessels or wells. The eluted samples may then be addressed by manual pipetting or by robotic transfer and separated by reverse phase chromatography and the separated sample constituents are analyzed by mass spectrometry.
In some embodiments, the biological sample may be digested with a protease.
Characteristic peptide fragments are produced by proteolysis, and the chosen signature peptides are measured as surrogate analytes for the anti-HCMV antibodies, In an exemplary embodiment, the biological sample may be digested with trypsin digestion. For trypsin digestion, samples may be reduced with DTT, S-carboxymethylated with sodium iodoacetate, and then digested with trypsin. Digested samples may be analyzed by a separation method, for example, reverse phase HPLC, e.g. Nucleosil CI8 column; size-exclusion chromatography (SEC), e.g, TSK
3000SWxL
column; or boronate affinity chromatography using a TSK Boronate column.
41.
Separation of Sample Constituents To form the analysis sample, the biological sample may be applied to a separation media to effect separation of more than one sample constituent. Separation methods include affinity, chromatography, and electrophoresis methods. Affinity methods include affinity chromatography, adsorption, and immobilized affinity matrices. Chromatography methods include HPLC, hydrophobic interaction (HIC), anion exchange, cation exchange, reverse-phase, normal phase, ion-pair reverse-phase, thin-layer, capillary flow, and size-exclusion.
Electrophoretic methods include single dimensional, slab gel, capillary, polyacrylamide, denaturing, native, free solution, paper, 2-dimensional, isoelectric focusing, and gradient voltage.
Other separation methods include: dialysis, centrifugation, sedimentation, floatation, precipitation, immunoprecipitatiort, and gel filtration.
Separation methods may effect separation of the constituents of the biological sample by one or more physico-chemical properties including, but not limited to, elution time, hydrophobicity, hydrophilicity, migration time, rate, velocity, chromatographic retention time, solubility, molecular volume or size, net charge, charge state, ionic charge, isoelectric point, dissociation constant (pKa), antibody affinity, electrophoretic mobility, ionization potential, dipole moment, hydrogen-bonding capability, and ion mobility in gas phase.
Low rate of flow by capillary flow infusion into the mass spectrometry inlet device facilitates sensitivity of mass detection, allowing for lower concentration analytes and higher molecular weight species such as intact proteins and antibodies to be detected and characterized.
Mass Spectrometry of Separated Sample Constituents Preparation of samples for mass spectrometric analysis can be conducted generally according to known techniques. See: "Modern Protein Chemistry: Practical Aspects", Howard, G. C. and Brown, W. E., Eds. (2002) CRC Press, Boca Raton, Florida.
The methods of the invention are appropriate for the analysis of antibody mixtures derived from biological samples where different chemical constituents of the mixture are first isolated, separated, or partially separated by one or more processes including affinity or chromatography which cause the constituents to elute sequentially or in a batch wise manner, or to be directly detected by mass spectrometry. Various structural features and properties of antibodies can be elucidated from mass spectrometry analysis including:
fragmentation, deamidation, glycation, oxidation, partial sequence information, e.g. N-terminal and C-terminal, dimer and aggregation states. One or more chemical constituents in the biological sample can be characterized in a highly specific manner by measurement of its accurate mass since the administered anti-HCMV antibody is of known sequence, structure, and molecular weight.
A variety of mass spectrometry systems capable of high mass accuracy, high sensitivity, and high resolution are known in the art and can be employed in the methods of the invention.
The mass analyzers of such mass spectrometers include, but are not limited to, quadrupole (Q), time of flight (TOF)õ ion trap, magnetic sector or FT-ICR or combinations thereof. The ion source of the mass spectrometer should yield mainly sample molecular ions, or pseudo-molecular ions, and certain characterizable fragment ions. Examples of such ion sources include atmospheric pressure ionization sources, e.g. electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and Matrix Assisted Laser Desorption Ionization (MALDI).
ESI and MALDI are the two most commonly employed methods to ionize proteins for mass 1.0 spectrometric analysis. ESI and APCI are the most commonly used ion source techniques for analysis of small molecules by LC/MS (Lee, M. "LC/MS Applications in Drug Development"
(2002) J. Wiley & Sons, New York), Surface Enhanced Laser 'Desorption Ionization (SELDI) is an example of a surface-based ionization technique that allows for high-throughput mass spectrometry (U.S.
Pat. No.
6,020,208). Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface such as a "protein chip" to interact with analytes, e.g., proteins, in solution. Such surfaces selectively interact with analytes and immobilize them thereon. Thus, the analytes of the invention can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing multiple reactive moieties at different sites on a substrate surface, throughput may be increased.
In functional systems, the mass spectrometer will accurately measure the mass of a.
chemical species of interest to within 20 ppm of its exact or calculated mass, and typically within 5 ppm or less of its exact or calculated mass. Commercially available mass analyzers can sample and record the whole mass spectrum simultaneously and with a frequency that allows enough spectra to be acquired for a plurality of constituents in the mixture to ensure that the mass spectrometric signal intensity or peak area is quantitatively representative.
This will also ensure that the elution times observed for all the masses would not be modified or distorted by the mass anal :,Ter and it would help ensure that quantitative measurements are not compromised by the need to measure abundances of transient signals.
Analytical variability may be corrected for by the use of an internal standard (IS) having physicochemical properties similar to that of the target analyte. (Mesmin et al. (2011) Bioanalysis 3: 477-480). In some embodiments, where signature peptides are measured as surrogate analytes for the anti-HCMV antibodies, stable isotope labled (SUL) peptides corresponding to the signature peptides may be used as internal standards.
(Hagman et al. (2008) Anal. Chem, 80: 1290-1296; Mesmin et al. (2010) Rapid Commun. Mass Spectrom, 24: 2875-2884).
Electrospray Ionization Mass Spectrometry (ES!) Higher sensitivity is achieved at lower flow rates due to increased analyte ionization efficiency (Gale et al (1993) Rapid Commun. Mass Spectrom. 7:1017). Thus by performing electrospray injection of a sample-containing fluid at flow rates in the nanoliter per minute range provides for accurate quantitation after proper calibration, and the high sensitivity for an analyte contained within the fluid when combined with mass spectrometry. Systems and devices including a miniaturized and consolidated micro-column and micro-column array having affinity chromatographic adsorbents, which offer high selectivity and sensitivity, and accurate qualitative analysis as front ends to MS have been reported (U.S. Pat. No, 6,811,689; U.S, Pat, No.
6,020,208; U.S. Pat. No. 6,579,719).
Masses of relatively high molecular weight compounds such as antibodies can be detected at mass-to-charge ratios (miz) that are easily determined by most mass spectrometers (typical miz ranges of up to 2000 to 3000). Electrospray ionization mass spectrometry ESI-MS, in particular, is suited for charged, polar or basic compounds and for analyzing multiply charged compounds with excellent detection limits. ES1 thus allows detection and characterization of large biomolecules, such as antibodies and antibody-drug conjugates with molecular weight (MW) of 150,000 or higher. With high-mass ions, a series of multiply charged molecular ions are typically observed. The molecular weight for positive ions is determined by multiplying the measured mlz ratio with the number of charges (n) minus the mass of the cation (C+) times the number of charges (n) on that ion, The ESI method allows the presence or absence of fragmentation to be controlled by controlling the interface lens potentials. Electrospray ionization (ES!) is compatible with liquid separation methods (front end), as well as mass spectrometric detection methods (back end) (Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications", Cole, R. B., Ed. (1997) Wiley, New York.
ESI-MS data may be acquired by averaging a number of scans together and smoothing the data to provide good peak intensity and shape. For low-mass compounds, the most abundant peaks observed are often the [M+H]+ ions in the positive-ion mode and [M-I-1]-in the negative ion mode. Doubly and triply charged ions as well as dimers may also be observed. Doubly charged positive ions will be observed at a mass (MW+2C+)/2 where MW is the molecular weight and C+ is the ionizing cation, such as HI, Nat, or NH4. Except for the very low mass compounds, the detected ions will be multiply charged. Due to the soft (low ionizing potential) conditions of EST, typically only molecular ions are observed. ESI spectra may have several molecular ion peaks that differ in the mass to charge ratio due to various numbers of charges the ion possesses, A dilute solution of a sample, e.g. ADC or other biomolecule may be slowly pumped through a hypodermic needle for EST-MS analysis, The sample may be introduced via flow injection or LC/MS. Typical flow rates range from less than 1 microliter (pi) per minute up to about one milliliter (ml) per minute, ESI is particularly suited for large biological molecules that are otherwise difficult to vaporize or ionize. The needle is held at a high voltage and the strong electric field at the end of the needle charges the nebulized solution and creates charged droplets.
The charged droplets evaporate water to ultimately yield molecular ions that travel into the vacuum chamber through a small orifice. During the process of solvent evaporation, the non-covalently bound complex is transferred from solution to gas phase, (Hu et al (1994)). Gentle desolvation conditions are generally required to maintain the intact gas-phase complex. The orifice may be heated to ensure that the ions are completely desolvated, Some MS systems may employ a counter-flowed heated gas. Charged droplets are emitted from a hypodermic needle and shrink as they evaporate solvent befOre entering a vacuum chamber. Heat and gas flows may be used to aid desolvation. The amount of sample required for EST measurements may be reduced by reducing the fluid flow by use of small capillary electrospray emitter, tips, a process known as nanoelectrospray. Natioelectrospra:,,,, methods can produce a constant sivial for about 10-30 minutes for a I pi sample. The low flow has been shown to increase the ion efficiency and reduce ion suppression. Nanoelectrospray methods are frequently used for MS/MS
protein studies (Korner et al (1996) J. Am, Soc. Mass Spectrom, 7:150-156; Mann, M.
and Wilm, M.
(1996) Anal, Chem. 68:1-8.
EST of proteins produce multiply charged ions with the number of charges tending to increase as the molecular weight increases. The number of charges on a given ionic species may be determined by methods such as: (1) comparing two charge states that differ by one charge and solving simultaneous equations; (ii) looking for species that have the same charge but different adduct masses; and (iii) examining the mass-to-charge ratios for resolved isotopic clusters, The methods of EST and ESI-MS and parameters needed to conduct these methods are well known in the art. The gentleness of the electrospray ionization process allows intact antibody conjugates to be directly detected by mass spectrometry.
In one embodiment, a Q1 mass spectrum of the protein, antibody, antibody fragment or antibody-conjugates (large molecules) is run as part of the method. A suitable quality Q1 mass spectrum of a large molecule can be obtained. Since there is potential for the protein envelope to shill, all the solvents used for chromatography are made fresh and acid is added to the elution solvent to position the spectrum envelop in the observed range. For proteins of greater than 100,000 mass units, an acid such as fOrmic acid can be used at about 0.1%
(volume) in the elution solvents, for example, both solvent A (water) and B (acetonitrile). A
stronger acid can be used, such as trifluoroacetic acid (TFA), at 0,05% (volume) TFA for both A and B solvents for proteins with less than 100,000 mass units. As the amount of formic acid is decreased, the intact glycosylated antibody, trastuzumab, picks up more charge, shifting the envelope further to the left and into the observed range of raiz (1800-3000 miz). As the declustering potential (DP) voltage is increased from about 30-120V to about 70-190V, the charge on the antibody increases even further. Thus voltage applied, solvent composition, and ion pairing agents are factors to consider and adjust. The declustering potential (DP) may be increased (ramped) to acquire enough resolution to select the best charge ion range. Linearity may be obtained over a wide range of mtz. Deglycosylation of the antibody assists quantitation of intact antibody or heavy chain, fragments or ADC. G1,2,:cosylation contributes to lower ionization efficiency and thus reduced sensitivity. When quantitating antibody or antibody fragment conjugates, cleglycosylation of the antibody may reduce the heterogeneity of the mass spectrum, increase sensitivity and thus simplifying the analysis.
Deconvolution tables are used to determine the exact mass to charge ratio (iniz) for each species to quantitated. Deconvolution software applications such as Analyst.TM, QS (Applied Biosystems, Foster City, Calif) are commercially available and/or provided with mass spectrometers. Deconvolution software generally provides the user with a table of deconvoluteti masses as well as a sub-table of miz ions used to calculate these masses.
E. Kits As a matter of convenience, the assay methods of this invention can be provided in the form of a kit. Such a kit is a packaged combination including the basic elements of (a) a capture reagent comprised of an anti-idiotypic antibody against the antibody of interest;
(b) a detectable (labeled or unlabeled) antibody that binds to the antibody of interest; and (c) instructions on how to perform the assay method using these reagents.
These basic elements are defined hereinabove.
The kit may further comprise a solid support for the capture reagents, which may be provided as a separate element or on which the capture reagents are already immobilized. Hence, the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit. In some embodiments, the capture reagents are coated on a microtiter plate. The detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies raised in a different species.
Where the label is an enzyme, the kit will ordinarily include substrates and cofactors required by the enzyme; where the label is a fluorophore, a dye precursor that provides the detectable chromophore; and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to 1-IRP or 0-galactosidase with MUG.
In various embodiments, the anti-idiotypic antibody is one or more of any of the anti-idiotypic antibodies disclosed herein. In some embodiments, the the anti-idiotypic antibody is selected from (a) a first anti-idiotypic antibody comprising the heavy chain sequence of SEQ ID
NO: 5 and the light chain sequence of SEQ ID NO: 7; (h) a second anti-idiotypic antibody comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO:
11; and (c) a combination thereof The kit also typically contains the antibody of interest as a standard (e.g., purified anti-C1 and/or anti-gH), as well as other additives such as stabilizers, washing and incubation buffers;
and the like.
The components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay.
Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
The following are examples of methods and compositions of the invention, It is understood that various other embodiments may be practiced, given the general description provided above.
Example 1: Generation of Anti Anti-CI and Anti-gil Hybridotnas Five Balble mice (Charles River Laboratories International, Inc., Wilmington, MA, USA) were hyperimmunized, in each hind footpad and intraperitoneally at 3-4 day intervals, with anti-CI or anti-g11 in an adjuvant containing metabolizable squalene, Tween 80, trehalose 6,6-dimycolate and monophosphoryl lipid A (all components obtained from Sigma Aldrich, USA).
47.
After 4 to 6 boosts, serum titers were evaluated by standard enzyme-linked immunosorbant assay (ELISA) to identify mice with positive serum titers to anti-CI or anti-gH. B
cells from spleens and popliteal lymph nodes were fused with mouse rnyeloma cells (X63.Ag80653 or P3X63Ag.U1; American Type Culture Collection, Manassas, VA, USA) by electrofusion (Hybrimune-Hybridoma Production System; Harvard Apparatus, Inc,, Holliston, MA, USA).
After 10-14 days, hybridoma supernatants were harvested and screened for CDR
specific antibody production by ELISA. All specific clones were then re-screened in a preliminary HCMIter PK. ELISA, in which candidate anti-ID materials were used as reagents to coat ELISA
plates and capture anti-C1 and anti-gH, and polyclonal anti-human antibodies were used as 1.0 detection reagents. Hybridoma clones 109E1.1, 4.25B10.15 and 2.41A2.4 showed high specific binding in the preliminary HCMV PK ELISAs after the first round of single cell per well subcloning (FACSAria cell sorter; BD Biosciences, San 'Jose, CA, USA), and therefore were scaled up (Inriova 2000 Shake Flask Format; New Brunswick Scientific, Enfield, CT, USA) for antibody production. The binding pattern of clone 2.41A2.4 in the PK assay suggested that this clone binds an epitope inclusive of the lambda light chain framework.
Supernatants were purified by affinity chromatography (MabSelect SuRe; GE Healthcare Bio-Sciences, Piscataway, NJ, USA), sterile-filtered, and stored at 4 C in PBS. Isotypes of the monoclonal antibodies were determined using the Isostrip Mouse rnAb Isotyping Kit (Roche Applied Biosciences, Indianapolis, IN, USA). The isotypes were determined to be IgGl, lambda (1.9E1.1), IgGl, kappa (4.251310.15) and IgG2a, kappa (2.41A2.4).
The binding affinities of the monoclonal antibodies were determined by Biacore analysis, as shown in Table 2.
Table 2 Monoclonal Antibody Antibody Bound Binding Affinity 4.251310.15 4.7 ni'vl 2.41A2.4 17 n_M
1.9E1.1 Anti-gH 0.1 nM
The amino acid sequences of the heavy and light chain variable domains for monoclonal antibodies 4,251310,15 and 1.9E1.1 were determined, as shown in Figures 1 and 2 for 4.251310.15, and in Figures 3 and 4 fer 1,9E1.1.
The hyper-variable regions of the heavy chain of monoclonal antibody 4.25B10.15 as shown in Figure 1 are:
HVR-H1 NYLIE SEQ ID NO: 13 HVR-H2 VINPGSGGTNYNEKFEA SEQ ID NO: 14 HVR-1-13 HGSSYWYFDV SEQ ID NO: 15 The hypervariable regions of the light chain of monoclonal antibody 4.25B10.15 as shown in Figure 2 are:
HVR-L1 RASQSISDYLH SEQ ID NO: 16 YASQSIS SEQ ID NO: 17 QNGHSFPYT SEQ. ID NO: 18 The hypervariable regions of the heavy chain of monoclonal antibody 109E1.1 as shown in Figure 3 are:
HVR-H1 DYWMY SEQ ID NO: 19 FIVR-H2 AIDTSDSYTTYNQNFKG SEQ ID NO: 20 HYR-H3 SGFFLFYYPMDY SEQ ID NO: 21 The hypervariable regions of the light chain of monoclonal antibody 1.9E1.1 as shown in Figure 4 are:
IIVR-L1 RSSTGAVTTSNYAN SEQ ID NO: 22 FIVR-L2 GTVNRAP SEQ ID NO: 23 FIVR-L3 ALWYSNHIN SEQ ID NO: 24 Example 2: ELISA Assay for Detection of Anti-g11 Figure 5 shows a schematic diagram of an ELISA assay used for detection of anti-gli (referred to as the "anti-gH clinical PK ELISA"). A biotin-conjugated anti-idiotypic antibody to ,anti-gH is bound to streptavidin-coated microplates and used to detect anti-gH in the sample.
The bound anti-gfi is detected using an HRP-conjugated mouse anti-human IgCi Peg antibody, Biotin-conjugated anti-idiotypic antibody is diluted at 500 lig/MIL in assay diluent (PBS/0.5% BSA/0.05% polysorbate 20/0.35 M NaC1/0.25% CHAPS/5 mIvI EDTA/0005 ,4 ProClin 300, pH 7.4 0.1). Roche SA plates are washed three times before use with wash buffer (PBS/0.05 /0 polysorbate 20, p1-17.46) before use. 50 of biotin-conjugated anti-idiotypic antibodies (1.9E1,1) is added to streptavidin-coated plates, and the plates are incubated for 25-35 minutes at room temperature with agitation. The curve standards, assay controls or samples are diluted 1:50 in assay diluent (described above). The diluted samples, curve standards and assay controls are added to the plates and incubated for 100-130 minutes at room temperature with agitation. The plates are washed four times. 1001AI/well of IIRP- conjugated mouse anti-huigG
Fey (8 ng/m1) is added, and the plates incubated for 1hr 5 minutes at room temperature with shaking. The plates are washed four times, and100 ul/well of TMB is added. The plates are incubated for 10-15 minutes at room temperature with agitation. 100 d of 1 M
phosphoric acid is added to each well. The plates are read using a 450 nm read wavelength and a 620 or 630 nm reference wavelength.
Figure 6 shows the binding activity of various anti-idiotypic antibodies to anti-C1 and anti-gH as tested in the preliminaay anti-HCMV human PK ELISA. The assay was carried out as follows: microliter ELISA plates were coated with anti-idiotypic monoclonal antibodies at 1 lag/int in PBS overnight. Anti-C1 and anti-gli were diluted in 1% NHS and added to the wells, being subsequently detected with 50 rigirriL (in-well concentration) of HRP-Sheep anti-huIgG.
As shown in Figure 6, only one anti-idiotypic antibody, 1.9E1.1, gave an optical density (01)) in the presence of anti-gli that was greater than two-fold the OD in the absence of anti-gH.
The other anti-idiotypic antibodies gave high background OD, suggesting cross-reaction of those anti-idiotypic antibodies with endogenous IgG in human serum (NHS).
The anti-gH clinical PK ELBA was used to quantify the amount of anti-gH spiked into sera from individual donors. Anti-gH was spiked at either high (2 lag/111W or low (0.3 p.g/mL) concentrations in neat serum. Figure 7 shows the percent recovery of individual serum spikes with anti-gli using 1.9E1.1 as the capture antibody. The assay Showed that anti-idiotypic antibody 1.9E1.1 provided good accuracy with all individual sera, spiked at both low and high anti-gH concentrations.
Example 3: ELBA Assay for Detection of Anti-CT
Figure 8 is a schematic diagram of an ELISA assay used for detection of anti-CI (referred to as the "anti-C1 clinical PK ELISA"). Anti-idiotypic antibody to anti-CI is bound to microplates and used to detect anti-CI in the sample. The bound anti-CI is detected using an :IMP-conjugated mouse anti-human IgG Fcg antibody, Microtiter plates are coated overnight at 2-8 C with 100 gl per well of 0.75 anti-C1 idiotypic antibody (in 0.05 M sodium carbonate buffer, pH9.6 0,1). The plates are washed three times with 400 l per well per cycle of wash buffer. 200 ul per well of blocking buffer (PBS/0.5% BSA/0M5% polysorbate 20/0.05% ProClin 300, pH 7,4 0.1) is added, and the plates are incubated at room temperature with shaking for 1-3 hours. Standard curves are prepared with standard/sample diluent (assay diluent with 0.5% normal pooled human serum;
assay diluent is composed of PBS/0.5 //0 BSA/0.05% polysorbate-20/0.35 M NaCl/0.25%
CHAPS/5inlvi EDTA/0.05% ProClin 300, pH 7.4 0,1). The plates are washed three times with 400 IA per well per cycle of wash buffer. Diluted standards, controls and samples are added to plates at 100 per well, in duplicate. The plates are incubated at room temperature with shaking for 2 hours 10 minutes. The plates are washed four times with 400 d per well per cycle of wash buffer. HRP
conjugates diluted in conjugate buffer (PBS10.54),43BSA/0.05% polysorbate 20/0.05% ProClin 300, pH 7.4 0,1) are added at 100 pi per well, The plates are incubated at room temperature with shaking for 1 hour 5 minutes, following which the plates are washed four times with 400 ul per well per cycle of wash buffer, 1001..d per well of TMB substrate is added, and the plates are incubated for approximately 15 minutes at room temperature with shaking. 100 ul of 1 M
phosphoric acid is added to each well. The plates are read using a 450 nni read wavelength and a 620 or 630 nm reference wavelength.
The anti-C1 clinical PK ELISA assay was carried out using plates coated with varying concentrations of capture antibody 4.25B10.15 in order to optimize the coating concentration. As shown in Table 3, plates coated with 0,75 mg/m1 of antibody 4.25B10.15 gave the optimum signal to background ratio, Background (bkgd) equals the assay signal obtained with unspiked human serum.
Table 3 Plate coat with 4.2510.15 (73427-34) coat Signalibkgd coat Signalibkgd coat Signalibkgd ' 8G8 (/m L) Iggimi. ratio 0.75tterral ratio a 5p,gimi. ratio 16 1.876 13.8 1.593 19.1 1.281 14.7 8 1.771 13.0 1.378 16.5 1.075 12.3 4 1,371 10,0 1,107 13.3 0.901 10.4 1 0.599 4.4 0.439 5,3 0.379 4,3 0.5 0.372 2.7 0.271 3.2 0,231 2.7 0.25 0.254 1,9 0.174 2.1 0.161 1.8 0.125 0.194 1.4 0.128 1.5 0.120 1.4 bkgd 0,136 0.084 0.087 The anti-CI clinical PK ELISA was run with a standard curve using anti-CI at varying concentrations spiked into 0.5% human serum pool. Results are summarized in Table 4. The assay showed a consistent dose response, and increasingly higher signal to background (SIB) ratio with increasing concentrations of anti-CI.
Table 4 Anti-Clugiml.. OD SiB ratio 20 1,970 22.3 10 1.725 19,6 5 1,203 13.6 2.5 0.743 8.4 1.25 0.431 4.9 0.625 0,261 3,0 0.313 0.174 2.0 0.156 0.134 1.5 bkgd 0.088 The anti-CI clinical PK ELISA was used to quantify the amount of anti-CI
spiked into sera from individual donors, using anti-idiotypic antibody 4.251310.15 or 4.2$F9.5 as the capture antibody. Anti-CI was spiked at either at 4 (high) or 0.5 (low) for the pool sample and samples 1-4; at either 12 p.whiL (high) or 0.6 pg/mL (low) for sample 5, and at either 7.62 li.g/mt (high) or 0,476 ggirn1_, (low) for samples 6 and 7. As Shown in Figure 9, aunti-idiotypic antibody 4.251310.15 performed better than 4,23F9,5, providing accurate spike recovery for all samples, at both high and low anti-CI concentrations.
Example 4: LC-MSINIS Assay for Quantification of Anti-CI and Anti-g11 in Human Serum An LC-MS/MS Assay was developed for quantification of anti-C1 and anti-gH in human serum. The assay used irnmunoaffinity capture to isolate two monoclonal antibodies, anti-CI and anti-gH, from human serum. Characteristic peptide fragments were then produced by proteolysis of the bound antibodies with trwsin, and the chosen signature peptides, along with their stable isotope-labeled internal standards (SIL ISs) were measured as surrogate analytes for anti-CI and anti-gH using HPLC with MS/MS detection. The signature tryptic peptides for anti-CI and anti-gH were as shown:
Anti-CI (P2) EQFVYVFOGGTK (SEQ ID NO: 25) Anti-CI (P4) DTSTSTAYLELSSLR (SEQ ID NO: 26) Anti-gH (P2) GLEWVSSINSNSR (SEQ ID NO: 27) Anti-gH (P5) LSC*AASOFTFSPYSVFWVR (SEQ ID NO: 28) Alkylated cysteine residue The SIL ISs comprised stable isotope-labeled amino acids at the amino acid positions shown in bold:
75 Anti-CI (P2) EQFVYVFGGGTK (SEQ ID NO: 25) Anti-CI (P4) DTSTSTAYLELSSLR (SEQ ID NO: 26) Anti-gH (P2) GLEWVSSINSNSR (SEQ ID NO: 27) Anti-gH (P5) LSC*AASGFTFSPYSVFWVR (SEQ ID NO: 28) Alkylated cysteine residue Signature peptides Anti-CI (P2) and Anti-gH (P2) are the primary surrogate peptides used for quantification of anti-CI and anti-gH, respectively, while Anti-CI (P4) and Anti-gH (P5) were secondary surrogate peptides used for quality control purposes. The method is applicable to the quantitation of anti-CI and anti-gH within a nominal range of 0,100 to 20,0 pg/mL, A 25-4 human serum aliquot is sufficient for analysis. Samples are stored in polypropylene tubes and kept frozen at approximately -70 'V prior to analysis.
The following sample types were extracted for evaluation purposes. Except where noted, the anti-idiotypic monoclonal antibody (anti-ID mAb) loads were 3.0 pig each per sample:
Pooled blank (MB) human serum.
Calibration Standards (CALs) fortified with anti-CI and anti-gli (combined) at the following concentrations .for each antibody: 0.100, 0.200, 0A00, LOO, 4.00, 8.00, 10,0 and 20.0 pig/mL (n=2 each level).
Quality Controls (QCs) fortified with anti-CI and anti-gH (combined) at the following concentrations for each antibody: 0.250, 2.00, 7.50 ug/m1., (n=4 each level).
Carryover blanks (CB) following high calibrators.
Over the curve dilution QC (DIL QC) prepared at anti-CI and anti-gH
concentration of 40 pg/mL and diluted 10-fold prior to aliquoting.
Specificity Samples (SP) from six individual human donors (n=1 each).
Fortified specificity samples (SPIE's) from six individual human donors fortified with anti-CI and anti-gH at a concentration of 0,200 pg/mL.
Additional Calibration Standards (CALs) fortified with anti-CI and anti-gH
(combined) at the following concentrations: 0.100, 0.200, 0.400, 1,00, 4,00, 8.00, 10.0 and 20.01.,ig/mL (n=2 each level) and containing anti-C1 anti-1D mAb and anti-gH anti-ID mAb loads of 1.50 jig each per sample.
The samples for analysis were processed by the following steps:
1. Precondition an Lmmulon 1B 96-well, flat-bottom inic,rotiter plate (Thermo, Product No. 3355) by adding 275 ul of plate-conditioning buffer (0.1:500:3.0:0,2:91.7:0.1 Tween 20 / Trizma HCI (1 M) NaCI (5 M) / ED'I'A (0.5 M)/ water / bovine serum albumin, v/v/v/v/w. Gently vortex the plate for approximately 1 min, Discard the conditioning buffer by inverting the plate, and tap plate dry on an absorbent pad.
2= A 25 jiL aliquot of serum sample is added to a mixture of 3.0 jig or 1.5 jig anti-1D mAb, 3.0 jig or 1.5 pig anti-gH anti-1D mAb and 10 mM FIBS-EP buffer (GE Healthcare, Product No, 94318) to yield a total volume of 1501,tL in the preconditioned 96-well micro-well plate.
3. Cover the plate with an adhesive film, and incubate at room temperature (RT) for 2 'vs under constant gentle shaking on a titer plate shaker.
4. The required volume of streptavidin Dynabeads M-280 (Life Technologies, Product No, 602-10) (based on 100 pit per well) is buffer exchanged with an equal volume of 10 mM HBS-EP by mixing at RT for 1-2 min. This procedure is repeated thrice.
After discarding the supernatant, the volume is made up with 10 mM HBS-EP to 50 pt so that the beads are concentrated 2-fold compared to the initial starting volume.
5. 50 III., of the concentrated beads are then added to each well from step 3 and allowed to bind at RT for 2 hrs under constant gentle shaking.
6.
Using an external magnet (Biotek 96-well Flat Magnet) and a Biotek plate washer, separate the magnetic beads and discard the unbound proteins in the supernatant. Wash the beads three times with 75 of bead washing buffer (5.0:3.0:0.2:91,8 Tiizman HC1 (1 M) /
NaCl (5 M) EDTA (0.5 M) / water, v/v/v/v) using the plate washer. Discard the supernatant after the beads are separated using the external magnet. Using the titer plate shaker, shake the titer plate between each of the three washes to resuspend the beads.
7. Wash the beads a fourth time with 200 pi, of bead washing buffer, discard the supernatant after the beads are separated using the external magnet, and shake briefly using the titer plate shaker to resuspend.
8. Separate the magnetic beads and discard the supernatant. Wash the beads one time with 75 p.IL of bead washing solution (20% acetonitrile in water), and and shake briefly using the titer plate shaker to resuspend. Separate the magnetic beads and discard the supernatant. Wash the beads a final time with bead washing solution, and and shake briefly using the titer plate shaker to resuspend.
9. After the washes are discarded, 75 RI, of Rapigest surfactant solution (0.05:40:10 Rapigest (Rapigest SF surfactant, Waters, Product No. 186002122) /Ammonium Bicarbonate, 50 mM / ACN, w/v/v), 25 L of internal standard (or internal standard diluent i.e.
20% acetonitrile in water), and 10 of DTT (0.1 M) is added to each well. The plate is incubated at 60 'C in a preheated oven for ¨1 hour.
20% acetonitrile in water), and 10 of DTT (0.1 M) is added to each well. The plate is incubated at 60 'C in a preheated oven for ¨1 hour.
10. Add 25 4, of iodoacettnide (0.1M) to each well, Mix well, Incubate at RT for hours (protected from light).
11. Add 2.5 ug of trypsin to each well. Mix well, Incubate at 37 'C
overnight (16-20 hrs).
overnight (16-20 hrs).
12. Add 15 ut of 2M HO to each well. Mix well. Incubate at 37 'C for ¨0,5 hours.
13. Place the plate on a magnet and transfer the entire solution to a Multicreen HTS
Filter Plate (Millipore, Part# MSHVN4550) placed on top of a 96-well Eppendorf Lo-bind collection plate.
Filter Plate (Millipore, Part# MSHVN4550) placed on top of a 96-well Eppendorf Lo-bind collection plate.
14. Centrifuge the filter plate/collection plate combination for 5 min at 3000 rpm to collect the filtrate.
15. Seal the plate with an injection mat, and directly inject 15 1.L (on a 10 injection loop) on the LC/MS/MS system.
Data Reduction The data system was configured to calculate and annotate the areas of MCMV3068A, MCMV5322A, and internal standard peaks automatically. A calibration curve was constructed using peak area ratios (PARs) of the calibration standards by applying a quadratic, 1/concentration squared weighted, least-squares regression algorithm. All concentrations were then calculated from their PARs against the calibration line, Initial experiments demonstrated that it was feasible to extend the calibration range of both analytes anti-CI and anti-gli from 0.100 to 20.0 uglmL for all signature peptides. A
quadratic regression (1/x2 weighted) was applied for anti-C1 and anti-gH (with a 1,5 pg each anti-ID rnAb load).
In addition, assay specificity was tested in multiple individual serum lots, and quality control (QC) samples were prepared and quantified against calibration standards. Good accuracy and precision data was obtained for the QCs for all four signature peptides.
Over-the-curve dilution QC samples were successfully diluted in the curve range. Consistent area ratios of the primary and the secondary peptides were observed for both analytes.
Specificity samples did not indicate any presence of interference at the analyte retention time. Fortified specificity samples did not indicate any significant evidence of lot-to-lot matrix effects.
The assay was further evaluated as follows, using anti -idiotypic antibody loads of 1.5 pg for each anti-i.diotypic antibody. Anti-idiotypic antibody 2.41A2.4 was used for detection of anti-CI, and anti-idiotypic antibody 1.9E1.1 was used for detection of anti-gH. Two sets of standard curves with at least eight calibration points ranging from 0.100 to 20.0 p.g/rnL of anti-C1 and anti-gH respectively were run; one curve at the beginning of the sample batch and one at the end, The back-calculated values of the calibration standards were required to be within *20% of the nominal concentration. If the back-calculated concentration of a standard fell outside the allowed range, that standard was excluded and the regression analysis of the calibration data repeated until all of the remaining values were within the allowed range. For an acceptable calibration curve, there were at least six calibration levels represented and a minimum of 75% of the total calibration standards in the run remaining following exclusions. At least one blank sample of each of the following types was analyzed: (a) reagent blank without internal standard; (b) matrix blank without internal standard; and (c) matrix blank with internal standard.
Lower limit of quantitation (LLOQ), Low, medium and high-level QC samples (n=6) were run to evaluate intra-assay precision and accuracy, As shown in Table 5, for the LLOQ
concentration of anti-C1 and anti-gH, the intra-assay coefficient of variation was less than 25,0%
(acceptance criterion) for the replicate i.ntra-assay determinations, and the mean accuracy was within 25,0% (acceptance criterion) of the theoretical analyte concentration. For all other concentrations of anti-CI and anti-gli, the intra-assay coefficient of variation was less than 20%
for the replicate intra-assay determinations, and the mean accuracy was within 20.0% of the theoretical analyte concentration. Thus the assay demonstrates acceptable precision and accuracy over a wide range of drug concentrations.
An additional "over the curve" matrix quality control pool was prepared with anti-CI and anti-gH concentration of approximately 600 p.g/mL, in order to assess the ability to dilute samples originally above the upper limit of the standard curve. Six replicates of this QC pool were individually diluted 100-fold using two serial 10-fold dilutions, and analyzed. The dilution was prepared using two serial I0-fold dilutions. As shown in Table 6, the intra-assay coefficient of variation was less than 20% for the replicate intra-assay determinations, and the mean accuracy was within 20.0% of the theoretical analyte concentration, Table 5 Antibody AitCl Anti.-gll Run ID IA-0 IA-1 IA-2 IA-3 IA-0 IA-1 IA-2 IA-(gagimL) ...............................................................
0,108 0.238 L60 16.8 0.0989 0.232 1,64 15.8 0,124 0.227 L65 17.3 0.109 0,229 1,44 13.3 ____________________ 0J05 0.221 1.70 15.7 0.120 0.210 1.70 14.9 0.109 0,222 1.66 16.4 0.108 0,225 1.75 13.3 0.103 0,192 1.62 14.6 0.111 0,225 1.26 13,4 0,105 0.211 1.72 16.1 0.116 0.217 1,50 15.5 ____________________ 6 6 6 6 6 6 Theoretical 0.100 0.200 1.50 15.0 0.100 0.200 1,50 15.0 Conceotratioo Mean 0,109 0.219 1.66 16.1 0.111 0.223 1,55 14.4 S.D.
0,0078 0.0157 0.047 0.930 0.0073 0,0082 0,185 1.17 % CN. .............. 7,18 7.17 2.85 5,76 6.55 3.67 11,9 8.1.6 % Difference 9.02 9,30 10.5 7,66 10.7 11,5 3.20 -4,18 from theoretical.
Low Limit 0.0750 0,160 1.20 12.0 0,0750 0.160 1.20 12.0 High Limit i 0.125 0,240 1.80 18.0 0,125 0.240 1.80 18.0 Table 6 Antibody Anti-CI Anti-gil Run ID QC 4 QC 4 (141.mi,) (..i,g/mL) 682 635 ..
Theoretical 600 600 Concentration Mean 639 664 37,4 66,7 % CAT, 5.85 10.1 % Difference 6.56 10.6 from theoretical Low Limit 480 480 High Limit 720 720 Matrix samples from at least ten different individual lots/donors (both unfortified (SP) and tbrtified with internal standard (SP/IS) only) were analyzed to evaluate assay specificity. For the unfortified specificity samples, the response of any interfering chromatographic background peak present at the expected retention time of an internal standard was less than 5% of the mean chromatographic response determined for that internal standard in the specificity samples fortified with that internal standard. For the specificity samples fortified with internal standard(s) only, the response ratio (interfering background peak response/internal standard peak response) measured in these samples was less than 20% of the mean response ratio determined for the corresponding analyte in the acceptable lower limit of quantification (LLOQ) CALs and QCs analyzed during the run, demonstrating acceptable assay specificity for both anti-CI and anti-gH.
Assay selectivity was evaluated by analysis of ten different individual lots fortified with the target analyte(s) at the low QC level and internal standard(s) at the level of use. These test samples are identified as SPF. The intra-assay coefficient of variation was less than 20% for the replicate determinations, and the mean accuracy was within 20.0% of the theoretical analyte concentration for all test samples of anti-C1 (Table 7) and for 9/10 test samples of anti-gH (Table 8), confirming acceptable assay selectivity for both antibodies.
Table 7 Sample ED SPF1 SPF 2 SPF 3 SPF 4 SPF 5 SPF 6 SPF 7 SPF 8 SPF9 SPF
0.189 0,157 0,215 0.182 0.184 1 0.182 0.167 0.178 0.218 0.186 0.171 0,193 0,204 0.183 0.224 0.182 0.178 0.166 0.185 0.194 0,211 0.217 0.228 0.192 0.150 0,204 0.220 0.186 0.190 0.202 Theoretical 0.200 0.200 0.200 0,200 0,200 0,200 0.200 0200. 0,200 0= .200 Omen Mean 0.190 0.189 0,216 0.186 0.186 0.189 0.188 0.177 0.198 0.194 S.D. 0,0199 0.0305 0,0121 0.0052 0.0370 0.0126 0.0278 0.0099 0.0175 + 0.0079 CN. 10.5 16.1 5.59 2.82 19.9 6.65 14.8 6.62 8.87 4,09 (.}.4 Difference -4.91 -5.52 7.76 -7.17 -7.02 -5.38 -5.92 -11,7 -1.23 -2.83 from theoretical ...............
Low Limit 0,160 0.160 0.160 0.160 0,160 0.160 0.160 0,160 0.160 0.160 High Limit 0.240 0.240 0.240 0,240 0,240 0,240 0,240 0.240 0.240 0.240 Table 8 Sample ID SPF 1 SPF 2 SPF 3 SPF 4 SPF 5 1 SPF 6 SPF 7 SPF 8 SPF 9 SPF
0.201 0.152 0.221 0.161 0.213 0.191 0.191 0.157 0,179 0.201 0.189 0.265 0.217 0.230 0.202 0.199 0.185 0.172 0.187 0.238 0.156 0.193 0,175 0.219 0.175 0.225 0.193 0.249 0.204 0= .204 Theoretical 0,200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0,200 Cone n Moan. 0.182 0.204 0.205 0,204 0,196 0,205 0,190 0.193 0,190 0= .214 S.D. 0.0231 0.0572 0.0255 0.0369 0,0194 0.0176 0.0045 0.0495 0,0127 0= .0207 % CV. 12,7 28.1 12.5 18.1 9.89 8.58 2.38 25,7 6,58 9.69 % Difference -9,03 1.78 2.34 1.78 -1.75 2.47 -5,17 -3,74 -5.05 7.00 from = theoretical Low Limit 0.160 0.160 0.160 0.160 0.160 0.160 0.160 0.160 0.160 0.160 High Limit 0.240 0,240 0.240 0.240 0.240 0.240 0.240 0.240 0.240 0,240 5 A cross-analyte interference cheek was evaluated for samples fortified with SIL ISs of signature peptides. Cross analyte interference evaluation between same individual analyte signature peptides was not evaluated for samples fortified with anti-CI or anti-gII, since the surrogate signature peptides corresponding to an individual analyte measured in this assay cannot be independently generated using the assay procedure. Rather, cross-analyte interference was evaluated between signature peptides corresponding to anti-el and signature peptides corresponding to anti-gH. Each internal standard was checked individually for possible contribution of signal to other analytes and internal standards. Control matrix samples ("Al") were fortified with only one internal standard at the expected level of use, and analyzed in triplicate. The contribution to the response of an analyte from a chromatographic peak present at its expected retention time was required to be less than 20% of the mean chromatographic response determined for that analyte in the acceptable LLOQ CALs and/or LLOQ
QCs analyzed during the run. The response of an interfering chromatographic background peak present at the expected retention time of an internal standard was required to be less than 5% of the mean chromatographic response determined for that internal standard in the acceptable upper limit of quantitation (LTLOQ) CALs and high-level QCs tbrtified with that internal standard and analyzed during the run. Overall, no significant cross-analyte interferences was observed for sample fortified with SIL ISs of signature peptides of anti-CI and anti-gli.
Cryofreezer freeze/thaw stability was evaluated at low and high QC
concentrations by subjecting the QCs to at least three freeze/thaw cycles. At least six replicates were analyzed per level. The coefficient of variation of freeze/thaw samples was less than or equal to 20% for replicate determinations, and the mean accuracy was within 20% of the theoretical concentration, demonstrating acceptable freeze/thaw stability for both anti-el and anti-gH.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference,
Data Reduction The data system was configured to calculate and annotate the areas of MCMV3068A, MCMV5322A, and internal standard peaks automatically. A calibration curve was constructed using peak area ratios (PARs) of the calibration standards by applying a quadratic, 1/concentration squared weighted, least-squares regression algorithm. All concentrations were then calculated from their PARs against the calibration line, Initial experiments demonstrated that it was feasible to extend the calibration range of both analytes anti-CI and anti-gli from 0.100 to 20.0 uglmL for all signature peptides. A
quadratic regression (1/x2 weighted) was applied for anti-C1 and anti-gH (with a 1,5 pg each anti-ID rnAb load).
In addition, assay specificity was tested in multiple individual serum lots, and quality control (QC) samples were prepared and quantified against calibration standards. Good accuracy and precision data was obtained for the QCs for all four signature peptides.
Over-the-curve dilution QC samples were successfully diluted in the curve range. Consistent area ratios of the primary and the secondary peptides were observed for both analytes.
Specificity samples did not indicate any presence of interference at the analyte retention time. Fortified specificity samples did not indicate any significant evidence of lot-to-lot matrix effects.
The assay was further evaluated as follows, using anti -idiotypic antibody loads of 1.5 pg for each anti-i.diotypic antibody. Anti-idiotypic antibody 2.41A2.4 was used for detection of anti-CI, and anti-idiotypic antibody 1.9E1.1 was used for detection of anti-gH. Two sets of standard curves with at least eight calibration points ranging from 0.100 to 20.0 p.g/rnL of anti-C1 and anti-gH respectively were run; one curve at the beginning of the sample batch and one at the end, The back-calculated values of the calibration standards were required to be within *20% of the nominal concentration. If the back-calculated concentration of a standard fell outside the allowed range, that standard was excluded and the regression analysis of the calibration data repeated until all of the remaining values were within the allowed range. For an acceptable calibration curve, there were at least six calibration levels represented and a minimum of 75% of the total calibration standards in the run remaining following exclusions. At least one blank sample of each of the following types was analyzed: (a) reagent blank without internal standard; (b) matrix blank without internal standard; and (c) matrix blank with internal standard.
Lower limit of quantitation (LLOQ), Low, medium and high-level QC samples (n=6) were run to evaluate intra-assay precision and accuracy, As shown in Table 5, for the LLOQ
concentration of anti-C1 and anti-gH, the intra-assay coefficient of variation was less than 25,0%
(acceptance criterion) for the replicate i.ntra-assay determinations, and the mean accuracy was within 25,0% (acceptance criterion) of the theoretical analyte concentration. For all other concentrations of anti-CI and anti-gli, the intra-assay coefficient of variation was less than 20%
for the replicate intra-assay determinations, and the mean accuracy was within 20.0% of the theoretical analyte concentration. Thus the assay demonstrates acceptable precision and accuracy over a wide range of drug concentrations.
An additional "over the curve" matrix quality control pool was prepared with anti-CI and anti-gH concentration of approximately 600 p.g/mL, in order to assess the ability to dilute samples originally above the upper limit of the standard curve. Six replicates of this QC pool were individually diluted 100-fold using two serial 10-fold dilutions, and analyzed. The dilution was prepared using two serial I0-fold dilutions. As shown in Table 6, the intra-assay coefficient of variation was less than 20% for the replicate intra-assay determinations, and the mean accuracy was within 20.0% of the theoretical analyte concentration, Table 5 Antibody AitCl Anti.-gll Run ID IA-0 IA-1 IA-2 IA-3 IA-0 IA-1 IA-2 IA-(gagimL) ...............................................................
0,108 0.238 L60 16.8 0.0989 0.232 1,64 15.8 0,124 0.227 L65 17.3 0.109 0,229 1,44 13.3 ____________________ 0J05 0.221 1.70 15.7 0.120 0.210 1.70 14.9 0.109 0,222 1.66 16.4 0.108 0,225 1.75 13.3 0.103 0,192 1.62 14.6 0.111 0,225 1.26 13,4 0,105 0.211 1.72 16.1 0.116 0.217 1,50 15.5 ____________________ 6 6 6 6 6 6 Theoretical 0.100 0.200 1.50 15.0 0.100 0.200 1,50 15.0 Conceotratioo Mean 0,109 0.219 1.66 16.1 0.111 0.223 1,55 14.4 S.D.
0,0078 0.0157 0.047 0.930 0.0073 0,0082 0,185 1.17 % CN. .............. 7,18 7.17 2.85 5,76 6.55 3.67 11,9 8.1.6 % Difference 9.02 9,30 10.5 7,66 10.7 11,5 3.20 -4,18 from theoretical.
Low Limit 0.0750 0,160 1.20 12.0 0,0750 0.160 1.20 12.0 High Limit i 0.125 0,240 1.80 18.0 0,125 0.240 1.80 18.0 Table 6 Antibody Anti-CI Anti-gil Run ID QC 4 QC 4 (141.mi,) (..i,g/mL) 682 635 ..
Theoretical 600 600 Concentration Mean 639 664 37,4 66,7 % CAT, 5.85 10.1 % Difference 6.56 10.6 from theoretical Low Limit 480 480 High Limit 720 720 Matrix samples from at least ten different individual lots/donors (both unfortified (SP) and tbrtified with internal standard (SP/IS) only) were analyzed to evaluate assay specificity. For the unfortified specificity samples, the response of any interfering chromatographic background peak present at the expected retention time of an internal standard was less than 5% of the mean chromatographic response determined for that internal standard in the specificity samples fortified with that internal standard. For the specificity samples fortified with internal standard(s) only, the response ratio (interfering background peak response/internal standard peak response) measured in these samples was less than 20% of the mean response ratio determined for the corresponding analyte in the acceptable lower limit of quantification (LLOQ) CALs and QCs analyzed during the run, demonstrating acceptable assay specificity for both anti-CI and anti-gH.
Assay selectivity was evaluated by analysis of ten different individual lots fortified with the target analyte(s) at the low QC level and internal standard(s) at the level of use. These test samples are identified as SPF. The intra-assay coefficient of variation was less than 20% for the replicate determinations, and the mean accuracy was within 20.0% of the theoretical analyte concentration for all test samples of anti-C1 (Table 7) and for 9/10 test samples of anti-gH (Table 8), confirming acceptable assay selectivity for both antibodies.
Table 7 Sample ED SPF1 SPF 2 SPF 3 SPF 4 SPF 5 SPF 6 SPF 7 SPF 8 SPF9 SPF
0.189 0,157 0,215 0.182 0.184 1 0.182 0.167 0.178 0.218 0.186 0.171 0,193 0,204 0.183 0.224 0.182 0.178 0.166 0.185 0.194 0,211 0.217 0.228 0.192 0.150 0,204 0.220 0.186 0.190 0.202 Theoretical 0.200 0.200 0.200 0,200 0,200 0,200 0.200 0200. 0,200 0= .200 Omen Mean 0.190 0.189 0,216 0.186 0.186 0.189 0.188 0.177 0.198 0.194 S.D. 0,0199 0.0305 0,0121 0.0052 0.0370 0.0126 0.0278 0.0099 0.0175 + 0.0079 CN. 10.5 16.1 5.59 2.82 19.9 6.65 14.8 6.62 8.87 4,09 (.}.4 Difference -4.91 -5.52 7.76 -7.17 -7.02 -5.38 -5.92 -11,7 -1.23 -2.83 from theoretical ...............
Low Limit 0,160 0.160 0.160 0.160 0,160 0.160 0.160 0,160 0.160 0.160 High Limit 0.240 0.240 0.240 0,240 0,240 0,240 0,240 0.240 0.240 0.240 Table 8 Sample ID SPF 1 SPF 2 SPF 3 SPF 4 SPF 5 1 SPF 6 SPF 7 SPF 8 SPF 9 SPF
0.201 0.152 0.221 0.161 0.213 0.191 0.191 0.157 0,179 0.201 0.189 0.265 0.217 0.230 0.202 0.199 0.185 0.172 0.187 0.238 0.156 0.193 0,175 0.219 0.175 0.225 0.193 0.249 0.204 0= .204 Theoretical 0,200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0,200 Cone n Moan. 0.182 0.204 0.205 0,204 0,196 0,205 0,190 0.193 0,190 0= .214 S.D. 0.0231 0.0572 0.0255 0.0369 0,0194 0.0176 0.0045 0.0495 0,0127 0= .0207 % CV. 12,7 28.1 12.5 18.1 9.89 8.58 2.38 25,7 6,58 9.69 % Difference -9,03 1.78 2.34 1.78 -1.75 2.47 -5,17 -3,74 -5.05 7.00 from = theoretical Low Limit 0.160 0.160 0.160 0.160 0.160 0.160 0.160 0.160 0.160 0.160 High Limit 0.240 0,240 0.240 0.240 0.240 0.240 0.240 0.240 0.240 0,240 5 A cross-analyte interference cheek was evaluated for samples fortified with SIL ISs of signature peptides. Cross analyte interference evaluation between same individual analyte signature peptides was not evaluated for samples fortified with anti-CI or anti-gII, since the surrogate signature peptides corresponding to an individual analyte measured in this assay cannot be independently generated using the assay procedure. Rather, cross-analyte interference was evaluated between signature peptides corresponding to anti-el and signature peptides corresponding to anti-gH. Each internal standard was checked individually for possible contribution of signal to other analytes and internal standards. Control matrix samples ("Al") were fortified with only one internal standard at the expected level of use, and analyzed in triplicate. The contribution to the response of an analyte from a chromatographic peak present at its expected retention time was required to be less than 20% of the mean chromatographic response determined for that analyte in the acceptable LLOQ CALs and/or LLOQ
QCs analyzed during the run. The response of an interfering chromatographic background peak present at the expected retention time of an internal standard was required to be less than 5% of the mean chromatographic response determined for that internal standard in the acceptable upper limit of quantitation (LTLOQ) CALs and high-level QCs tbrtified with that internal standard and analyzed during the run. Overall, no significant cross-analyte interferences was observed for sample fortified with SIL ISs of signature peptides of anti-CI and anti-gli.
Cryofreezer freeze/thaw stability was evaluated at low and high QC
concentrations by subjecting the QCs to at least three freeze/thaw cycles. At least six replicates were analyzed per level. The coefficient of variation of freeze/thaw samples was less than or equal to 20% for replicate determinations, and the mean accuracy was within 20% of the theoretical concentration, demonstrating acceptable freeze/thaw stability for both anti-el and anti-gH.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference,
Claims (52)
1. An isolated anti-idiotypic antibody that specifically binds to an anti-HCMV
antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 or an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO:
3 and the light chain sequence of SEQ ID NO: 4,
antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 or an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO:
3 and the light chain sequence of SEQ ID NO: 4,
2. The anti-idotypic antibody of claim 1, wherein the anti-idotypic antibody specifically binds to an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID
NO: 1 and the light chain sequence of SEQ ID NO: 2.
NO: 1 and the light chain sequence of SEQ ID NO: 2.
3. The anti-idotypic antibody of claim 1, wherein the anti-idotypic antibody specifically binds to an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID
NO: 3 and the light chain sequence of SEQ ID NO: 4.
NO: 3 and the light chain sequence of SEQ ID NO: 4.
4. The anti-idiotypic antibody of claim 2, wherein the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein:
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ :ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ :ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
5, The and-idiotypic antibody of claim 4, wherein the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID
NO: 7,
NO: 7,
6. The anti-idiotypic antibody of claim 3, wherein the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-1, HVR-L2 and HVR-L3), wherein:
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
7. The anti-idiotypic antibody of claim 6, wherein the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID
NO: 11.
NO: 11.
8. The anti-idiotypic antibody of claim 2, wherein the anti-idiotypic antibody specifically binds to at le.ast one HVR, of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2.
9, The anti-idiotypic antibody of claim 3, wherein the anti-idiotypic antibody specifically binds to at least one HVR of an anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO; 3 and the light chain sequence of SEQ ID NO: 4.
10. The anfi-idiotypic antibody of clairn 3, wherein the anti-idiotypic antibody specifically binds to HVR-H2 (SEQ ID NO: 36) of an anti-.HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 3 and the light chain sequence of SEQ ID
NO: 4.
NO: 4.
11. The a.nti-idiotypic antibody of claim I conjugated to a detectable label.
12. The antibody of claim I conjugated to biotin,
13. An enzyme-linked immunosorbent assay (ELISA) method for specifically detecting in a biological sarnple an antibody of interest comprising (a) contacting and incubating the biological sample with a capture reagent, wherein the capture reagent is the anti-idiotypic antibody of claim 1, so as to bind any of the antibody of interest present in the sample., and (h) contacting the capture reagent, and hence any bound antibody of interest, with a detectable antibody that binds to the antibody of interest, and measuring the level of the antibody of interest bound to the anti-idiotypic antibody using a detection means for the detectable antibody, wherein the antibody of interest is selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2;
(b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
(b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
14. The method of claim 13, wherein the capture reagent is immobilized to a solid support and the method further comprises the step of separating the biological sample from the immobilized capture reagent bound to any of the antibody of interest present.
15. The method of claim 14, wherein the immobilized capture reagent is coated on a microtiter plate.
16. The method of claim 14, wherein the immobilized capture reagent is conjugated to biotin and bound to a streptavidin coated microtiter plate.
17. The method of claim 16, wherein the detectable antibody is an antibody from a non-human species that binds to human antibodies.
18. The method of claim 17, wherein the detectable antibody is a mouse anti-huIgG
Fc.gamma. antibody.
Fc.gamma. antibody.
19. The method of claims 17 or 18, wherein the detectable antibody is directly detectable.
20. The method of claims 17 or 18, wherein the detectable antibody is conjugated to horseradish peroxidase.
21. The method of claims 17 or 18, wherein the detectable antibody is detected by a fluorimetric or calorimetric reagent.
22. A method for specifically detecting in a biological sample an antibody of interest comprising; (a) contacting and incubating the biological sample with an anti-idiotypic antibody that specifically binds to the antibody of interest; (b) contacting and incubating the sample with immunoaffinity beads that bind to the anti-idiotypic antibody; (c) eluting the antibody of interest;
(d) applying the eluted antibody of interest to a separation media to effect separation of more than one sample constituent wherein a separated sample constituent comprises antibody of interest or a fragment or signature peptide thereof; and (e) establishing the mass to charge ratio of one or more separated sample constituents by mass spectrometry, wherein the antibody of interest is selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1. and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO:
4; and (c) a combination thereof.
(d) applying the eluted antibody of interest to a separation media to effect separation of more than one sample constituent wherein a separated sample constituent comprises antibody of interest or a fragment or signature peptide thereof; and (e) establishing the mass to charge ratio of one or more separated sample constituents by mass spectrometry, wherein the antibody of interest is selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1. and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO:
4; and (c) a combination thereof.
23: The method of claim 22 further comprising treating the biological sample with a protease after incubation with the immunoaffinity beads and prior to or after eluting the antibody of interest.
24. The method of claim 23 wherein the protease is trypsin.
25. The method of claim 22 wherein the anti-idiotypic antibody is biotinylated,
26. The method of claim 25 wherein the anti-idiotypic antibody binds to streptavidin coated paramagnetic immunoaffinity beads.
27. The method of claim 26 wherein the anti-idiotypic antibodies are bound to the immunoaffinity beads prior to contact and incubation with the biological sample.
28. The method of claim 22 wherein the immunoaffinity bead is a magnetic bead.
29. The method of claim 22 wherein the separation media is a chromatography support.
30. The method of any one of claims 13-18 and 22-29, wherein the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ
ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7.
31: The method of any one of claims 1 3-1 8 and 22-29, wherein the antibody of interest is an anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2 and the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3) and three tight chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein:
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
32. The method of any one of claims 13-18 and 22-29, wherein the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ NO: 7.
33. The method of any one of claims 13-18 and 22-29, wherein the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ
ID NO: 3 and the light chain sequence of SEQ ID NO: 4 and the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 11.
ID NO: 3 and the light chain sequence of SEQ ID NO: 4 and the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 11.
34. The method of any one of claims 13-18 and 22-29, wherein the antibody of interest is an anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4 and the anti-idiotypic antibody comprises three heavy chain hypervariable regions (HVR-H1,HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein:
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ IT) NO: 24,
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ IT) NO: 24,
35. The method of any one of claims 13-18 and 22-29, wherein the anti-idiotypic antibody comprises the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ NO: 11.
36. The method of any one of claims 13-18 and 22-29, wherein the antibody of interest is an anti-HCMV antibody comprising the heavy chain sequence of SEQ
ID NO: 3 and the light chain sequence of SEQ ID NO: 4 and the anti-idiotypic antibody specifically binds to HVR-H2 (SEQ ID NO: 36) of said anti-HCMV antibody.
ID NO: 3 and the light chain sequence of SEQ ID NO: 4 and the anti-idiotypic antibody specifically binds to HVR-H2 (SEQ ID NO: 36) of said anti-HCMV antibody.
37. The method of any one of claims 13-18 and 22-29, wherein the antibody of interest is a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2 and (b) a second anti-HCMV
antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID
NO: 4.
antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID
NO: 4.
38. The method of any one of claims 13-18 and 22-29, wherein the anti-idiotypic antibody binds to the antibody of interest and not to at least one other anti-HCMV antibody in the sample.
39. The method of any one of claims 13-18 and 22-29, wherein the biological sample is isolated from a human subject.
40. The method of claim 39, wherein the human subject has been treated with an anti-HCMV antibody selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ 1 NO: 1 and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy Chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO: 4; and (c) a combination thereof.
41. The method of any one of claims 13-18, 22-29, and 40, wherein the method further comprises using a standard curve to determine the level of the antibody of interest compared to a known level.
42. The method of any one of claims 13-18, 22-29, and 40, wherein the biological sample is blood, plasma or serum.
43. The method of claim 41, wherein the sample is serum.
44. An immunoassay kit for specifically detecting in a biological sample an antibody of interest selected from (a) a first anti-HCMV antibody comprising the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2; (b) a second anti-HCMV antibody comprising a heavy chain sequence of SEQ ID NO: 3 and a light chain sequence of SEQ ID NO:
4; and (c) a combination thereof the kit comprising: (a) a container containing, as a capture reagent, an anti-idiotypic antibody that specifically binds to the antibody of interest; (b) a container containing a detectable antibody that binds to the antibody of interest; and (c) instructions for detecting said antibody of interest.
4; and (c) a combination thereof the kit comprising: (a) a container containing, as a capture reagent, an anti-idiotypic antibody that specifically binds to the antibody of interest; (b) a container containing a detectable antibody that binds to the antibody of interest; and (c) instructions for detecting said antibody of interest.
45. The kit of claim 44 useful in an ELISA method for detecting the antibody of interest.
46. The kit of claim 45 further comprising a solid support for the capture reagent.
47. The kit of claim 46, wherein the capture reagent is immobilized on the solid support.
48. The kit of claim 47, wherein the capture reagent is coated on a microtiter plate.
49. The kit of claim 44, wherein the anti-idiotypic antibody is selected from (a) a first anti-idiotypic antibody comprising the heavy chain sequence of SEQ ID NO: 5 and the light chain sequence of SEQ ID NO: 7; (b) a second anti-idiotypic antibody comprising a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 11; and (c) a combination thereof.
50. An isolated anti-idiotypic antibody that specifically binds to an anti-HCMV
antibody comprising at least one heavy chain hypervariable region selected from the group consisting of NO: 13 - 24.
antibody comprising at least one heavy chain hypervariable region selected from the group consisting of NO: 13 - 24.
51. The antibody of claim 50 comprising three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein:
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 13;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 14;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 15;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 16;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18.
52.
The antibody of claim 50 comprising three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein:
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
The antibody of claim 50 comprising three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3) and three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3), wherein:
(a) HVR-H1 comprises the amino acid sequence of SEQ ID NO: 19;
(b) HVR-H2 comprises the amino acid sequence of SEQ ID NO: 20;
(c) HVR-H3 comprises the amino acid sequence of SEQ ID NO: 21;
(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO: 22;
(e) HVR-L2 comprises the amino acid sequence of SEQ ID NO: 23; and (f) HVR-L3 comprises the amino acid sequence of SEQ ID NO: 24.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261616914P | 2012-03-28 | 2012-03-28 | |
US61/616,914 | 2012-03-28 | ||
PCT/US2013/032661 WO2013148373A1 (en) | 2012-03-28 | 2013-03-15 | Anti-hcmv idiotypic antibodies and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2868161A1 true CA2868161A1 (en) | 2013-10-03 |
Family
ID=48087712
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2868161 Abandoned CA2868161A1 (en) | 2012-03-28 | 2013-03-15 | Anti-hcmv idiotypic antibodies and uses thereof |
Country Status (12)
Country | Link |
---|---|
US (2) | US9139659B2 (en) |
EP (1) | EP2831119A1 (en) |
JP (1) | JP2015518471A (en) |
KR (1) | KR20140138971A (en) |
CN (1) | CN104334583A (en) |
AR (1) | AR090510A1 (en) |
BR (1) | BR112014024023A2 (en) |
CA (1) | CA2868161A1 (en) |
HK (1) | HK1206759A1 (en) |
MX (1) | MX2014011646A (en) |
RU (1) | RU2014143251A (en) |
WO (1) | WO2013148373A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2015534959A (en) * | 2012-10-19 | 2015-12-07 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Inhibitor of SYK |
EP3559044A4 (en) | 2016-12-23 | 2020-12-02 | REMD Biotherapeutics, Inc. | Immunotherapy using antibodies that bind programmed death 1 (pd-1) |
KR20190104411A (en) * | 2017-01-18 | 2019-09-09 | 제넨테크, 인크. | Idiotype Antibodies to Anti-PD-L1 Antibodies and Uses thereof |
WO2018222675A1 (en) * | 2017-05-30 | 2018-12-06 | The Board Of Regents Of The University Of Oklahoma | Anti-doublecortin-like kinase 1 antibodies and methods of use |
CN112899235A (en) * | 2019-12-03 | 2021-06-04 | 信达生物制药(苏州)有限公司 | Cell culture solution harvesting method |
WO2022251311A1 (en) * | 2021-05-25 | 2022-12-01 | Seagen Inc. | Methods of quantifying anti-cd40 antibodies |
Family Cites Families (34)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SE337223B (en) | 1967-05-23 | 1971-08-02 | Pharmacia Ab | |
US3720760A (en) * | 1968-09-06 | 1973-03-13 | Pharmacia Ab | Method for determining the presence of reagin-immunoglobulins(reagin-ig)directed against certain allergens,in aqueous samples |
US3645090A (en) | 1969-06-19 | 1972-02-29 | Citizen Watch Co Ltd | Day-date quick-adjuster for calender timepiece |
US3691016A (en) | 1970-04-17 | 1972-09-12 | Monsanto Co | Process for the preparation of insoluble enzymes |
US3940475A (en) | 1970-06-11 | 1976-02-24 | Biological Developments, Inc. | Radioimmune method of assaying quantitatively for a hapten |
CA1023287A (en) * | 1972-12-08 | 1977-12-27 | Boehringer Mannheim G.M.B.H. | Process for the preparation of carrier-bound proteins |
US4195128A (en) | 1976-05-03 | 1980-03-25 | Bayer Aktiengesellschaft | Polymeric carrier bound ligands |
US4330440A (en) | 1977-02-08 | 1982-05-18 | Development Finance Corporation Of New Zealand | Activated matrix and method of activation |
CA1093991A (en) | 1977-02-17 | 1981-01-20 | Hideo Hirohara | Enzyme immobilization with pullulan gel |
US4229537A (en) | 1978-02-09 | 1980-10-21 | New York University | Preparation of trichloro-s-triazine activated supports for coupling ligands |
US4376110A (en) | 1980-08-04 | 1983-03-08 | Hybritech, Incorporated | Immunometric assays using monoclonal antibodies |
US4536479A (en) * | 1983-03-22 | 1985-08-20 | E. I. Du Pont De Nemours And Company | Use of anti-idiotype antibodies in immunoassays |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4737456A (en) | 1985-05-09 | 1988-04-12 | Syntex (U.S.A.) Inc. | Reducing interference in ligand-receptor binding assays |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
EP0564531B1 (en) | 1990-12-03 | 1998-03-25 | Genentech, Inc. | Enrichment method for variant proteins with altered binding properties |
WO1993006217A1 (en) | 1991-09-19 | 1993-04-01 | Genentech, Inc. | EXPRESSION IN E. COLI OF ANTIBODY FRAGMENTS HAVING AT LEAST A CYSTEINE PRESENT AS A FREE THIOL, USE FOR THE PRODUCTION OF BIFUNCTIONAL F(ab')2 ANTIBODIES |
US6020208A (en) | 1994-05-27 | 2000-02-01 | Baylor College Of Medicine | Systems for surface-enhanced affinity capture for desorption and detection of analytes |
US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
NZ516848A (en) | 1997-06-20 | 2004-03-26 | Ciphergen Biosystems Inc | Retentate chromatography apparatus with applications in biology and medicine |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
AU782626B2 (en) | 1999-10-04 | 2005-08-18 | Medicago Inc. | Method for regulating transcription of foreign genes |
US20020139751A1 (en) | 2001-02-20 | 2002-10-03 | Sheng Zhang | Microchip electrospray device and column with affinity adsorbents and use of the same |
JP2007528868A (en) * | 2003-08-14 | 2007-10-18 | ワイス | Anti-Lewis Y anti-idiotype antibody and use thereof |
US7704510B2 (en) * | 2006-06-07 | 2010-04-27 | The Trustees Of Princeton University | Cytomegalovirus surface protein complex for use in vaccines and as a drug target |
AU2007331463B2 (en) | 2006-12-15 | 2014-03-27 | Ribovax Biotechnologies Sa | Antibodies against human cytomegalovirus (hCMV) |
US7947274B2 (en) | 2007-01-04 | 2011-05-24 | Humabs, LLC. | Human cytomegalovirus neutralising antibodies and use thereof |
GB0700133D0 (en) | 2007-01-04 | 2007-02-14 | Humabs Llc | Human cytomegalovirus neutralising antibodies and use thereof |
CN102203133B (en) * | 2008-07-16 | 2015-01-07 | 生物医学研究学会 | Human cytomegalovirus neutralizing antibodies and use thereof |
CN102892782B (en) * | 2009-12-23 | 2015-01-14 | 4-抗体股份公司 | Binding members for human cytomegalovirus |
WO2012047732A2 (en) * | 2010-09-29 | 2012-04-12 | Genentech, Inc. | Antibody compositions and methods of use |
EP2793941A1 (en) * | 2011-12-23 | 2014-10-29 | F.Hoffmann-La Roche Ag | Articles of manufacture and methods for co-administration of antibodies |
-
2013
- 2013-03-15 EP EP13715815.0A patent/EP2831119A1/en not_active Withdrawn
- 2013-03-15 RU RU2014143251A patent/RU2014143251A/en not_active Application Discontinuation
- 2013-03-15 BR BR112014024023A patent/BR112014024023A2/en not_active IP Right Cessation
- 2013-03-15 CA CA 2868161 patent/CA2868161A1/en not_active Abandoned
- 2013-03-15 MX MX2014011646A patent/MX2014011646A/en unknown
- 2013-03-15 CN CN201380027593.4A patent/CN104334583A/en active Pending
- 2013-03-15 US US13/841,691 patent/US9139659B2/en not_active Expired - Fee Related
- 2013-03-15 WO PCT/US2013/032661 patent/WO2013148373A1/en active Application Filing
- 2013-03-15 KR KR1020147029028A patent/KR20140138971A/en not_active Application Discontinuation
- 2013-03-15 JP JP2015503347A patent/JP2015518471A/en active Pending
- 2013-03-26 AR ARP130100989 patent/AR090510A1/en unknown
-
2015
- 2015-08-03 HK HK15107419.1A patent/HK1206759A1/en unknown
- 2015-09-21 US US14/860,344 patent/US20160002355A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP2831119A1 (en) | 2015-02-04 |
KR20140138971A (en) | 2014-12-04 |
WO2013148373A8 (en) | 2014-09-25 |
US20130266973A1 (en) | 2013-10-10 |
US20160002355A1 (en) | 2016-01-07 |
AR090510A1 (en) | 2014-11-19 |
WO2013148373A1 (en) | 2013-10-03 |
CN104334583A (en) | 2015-02-04 |
MX2014011646A (en) | 2015-02-24 |
HK1206759A1 (en) | 2016-01-15 |
JP2015518471A (en) | 2015-07-02 |
RU2014143251A (en) | 2016-05-20 |
BR112014024023A2 (en) | 2017-07-18 |
US9139659B2 (en) | 2015-09-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11279762B2 (en) | Idiotypic antibodies against anti-PD-L1 antibodies and uses thereof | |
US20160002355A1 (en) | Idiotypic antibodies and uses thereof | |
US20210165000A1 (en) | Methods For Detecting Phosphorylated Alpha-Synuclein | |
CN112105640A (en) | Assays for detecting neurodegeneration | |
US20210047431A1 (en) | Anti-pla2-gib antibodies and the uses thereof | |
US20210040236A1 (en) | Substances and methods for the use in prevention and/or treatment in huntington's disease | |
US20210382067A1 (en) | High-throughput and mass-spectrometry-based method for quantitating antibodies | |
CN115298215A (en) | Specifically binds to primary immunodeficiency: antibodies to peptides related to viskott-aldrich syndrome and X-linked agammaglobulinemia | |
EP3665203B1 (en) | Method for determining anti-drug antibodies in a minipig sample | |
US11560421B2 (en) | Broad-spectrum monoclonal antibodies against chikungunya virus E1 structural protein | |
US20230228763A1 (en) | Process for direct readout of immunoglobulins | |
KR20220092744A (en) | Monoclonal Antibody specific for Equine influenza virus H3N8 and Composition for detecting Equine influenza virus using the same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |
Effective date: 20180315 |